Glycosaminoglycan Compositions in Combination with Stem Cells

ABSTRACT

A pharmaceutical preparation for treating connective tissue damage in man and in animals, comprising a therapeutically effective amount of a glycosaminoglycan composition comprising chondroitin sulfate, N-acetyl D-glucosamine, and hyaluronan, in combination with isolated stem cells. Methods of use and kits containing the glycosaminoglycan composition and materials for isolating stem cells and for treating connective tissue damage and repair of cartilage in man and in animals are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation in part of U.S. Ser. No.11/766,510 filed Jun. 21, 2007, pending, which is incorporated herein byreference in its entirety.

FIELD OF INVENTION

The present invention is generally directed to a pharmaceuticalpreparation, and methods of use thereof, for treating connective tissuedamage and repairing cartilage in man and in animals. More particularly,the present invention provides a pharmaceutical preparation comprising aglycosaminoglycan composition comprising one or more glucosaminoglycans(GAGs) in combination with stem cells or a pharmaceutically acceptableformulation comprising stem cells for use in treating connective tissuedisorders and repairing cartilages.

BACKGROUND OF THE INVENTION

The four primary types of vertebrate tissues are epithelial tissue,muscle tissue, nerve tissue, and connective tissue. Connective tissuesare typically involved in structure and support, and are usually derivedfrom mesoderm cells. Connective tissue is widespread in the body, and asthe name implies, it primarily serves a connecting function to bind orstrengthen organs or other tissues. It also functions inside the body todivide and compartmentalize other tissue structures.

In vertebrates, the most common type of connective tissue is looseconnective tissue. Loose connective tissue holds organs in place andattaches epithelial tissue to other underlying tissues. Loose connectivetissue is named based on the “weave” of its constituent fibers. Thereare three main component types of loose connective tissue: collagenousfibers, elastic fibers, and reticular fibers. Collagenous fibers aremade of collagen and consist of bundles of fibrils that are coils ofcollagen molecules. Elastic fibers are made of elastin and arestretchable. Reticular fibers join connective tissues to other tissues.Loose connective tissue also includes adipose tissue that stores fat.

Another type of connective tissue is fibrous connective tissue, which isfound in tendons and ligaments. Fibrous connective tissue is composed oflarge amounts of closely packed collagenous fibers. Cartilage is a formof fibrous connective tissue that is composed of closely packedcollagenous fibers in a rubbery gelatinous substance called chondrin.The skeletons of sharks are composed of cartilage. Cartilage alsoprovides flexible support for certain structures in humans including thenose, trachea, ears, and articulating joints, for example.

Bone and blood are two other specialized connective tissues. Bone is atype of mineralized connective tissue that contains collagen and calciumphosphate, a mineral crystal. Calcium phosphate gives bone its firmness.Blood is also considered a type of connective tissue. Even though it hasa different function in comparison to other connective tissues it doeshave an extracellular matrix. The matrix is the plasma and erythrocytes,leukocytes and platelets are suspended in the plasma.

The connective tissues of humans and animals are constantly subjected tostresses and strains from mechanical forces and from diseases that canresult in afflictions, such as arthritis, joint inflammation stiffnessand connective tissue injuries such as tendonitis, bursitis, strained ortorn ligaments and tendons and the like. Indeed, connective tissueafflictions are quite common, presently affecting millions of people andanimals. Further, such afflictions can be not only painful but, in theirextreme, debilitating.

Arthritic diseases, characterized by pain, inflammation and stiffness ofthe joints leading to reduced range of mobility, are due to thedegradation of connective tissue (mainly cartilage) in joints. Suchdiseases particularly affect weight-bearing joints such as the hips,knees, spine, ankles and feet and those joints with frequent movementsuch as hands, arms and neck. For instance, osteoarthritis (OA) inparticular is a degenerative disease of the joint cartilage resulting innarrowing of the joint space and changes in the underlying bone(Barclay, et al., The Annals of Pharmacotherapy, (May, 1998) 32:574-79). Osteoarthritis is the most common form of arthritis and itaffects approximately one in ten people in North America. Osteoarthritisis not limited to humans, but occurs in other mammals such as horses,dogs, cats, mice and guinea pigs as well, making osteoarthritis one ofthe most common sources of chronic pain seen by veterinarians.

Rheumatoid arthritis (RA) is a connective tissue disease that has somesimilar symptoms to osteoarthritis. Rheumatoid arthritis is among themost debilitating of all forms of arthritis, causing joints to ache andthrob and eventually become deformed. Sometimes these symptoms make eventhe simplest daily activities difficult to manage. The exact cause ofrheumatoid arthritis is unknown, however, it is believed to be anautoimmune disease (Maini, et al., Aetiopathogenesis of RheumatoidArthritis. in Mechanisms and Modes of Rheumatoid Arthritis, (1995)Academic Press Ltd. pp. 25-46), in which the immune system attacks bodytissues, e.g., the synovium, as if they were foreign invaders,culminating in inflammatory and destructive responses in joints as wellas other tissues. It has also been postulated that rheumatoid arthritisis triggered by an infection, possibly a virus or bacterium in peoplewith an inherited susceptibility. Some researchers also believe thathormones may be involved in the development of rheumatoid arthritis.

As with some other forms of arthritis, rheumatoid arthritis involvesinflammation of the joints. In rheumatoid arthritis, white blood cells,whose usual job is to attack unwanted invaders, such as bacteria andviruses, move from the bloodstream into the synovium. Here, these bloodcells appear to play an important role in causing the synovial membraneto become inflamed (synovitis). This inflammation results in the releaseof proteins that, over months or years, cause thickening of thesynovium. These proteins can also damage cartilage, bone, tendons andligaments. Gradually, the joint loses its shape and alignment andeventually, it may be destroyed.

Under normal conditions, the body maintains the synovial joint in stateof homeostasis through a variety of complex hormonal and mechanicalfeedback mechanisms. Several types of insult or injury can upset thedelicate homeostatic balance. For example, repeated trauma or stress(slow chronic insult) to the joint during everyday use, e.g., athletictraining or performance, is often the inciting cause of jointinflammation and loss of homeostasis. Initially, such stress results inonly soft tissue inflammation in the form of synovitis or capsulitis(e.g., traumatic synovitis). Cartilage damage may or may not initiallybe present in the early stages of stress related injury or inflammation.However, the release of inflammatory mediators into the joint such asprostaglandins, cytokines, lysosomal enzymes and free radicals can leadto damage of articular cartilage and can cause cartilage degradation andleading to development of degenerative joint disease (DJD).

A second type of insult or injury, the osteochondral defect, e.g., achip fracture, is often associated with an acute mechanical failure ortraumatic injury, e.g., an acute racing or training injury, although,such a fracture can be due to secondary complications associated withchronic DJD. Under this scenario, the lesion often starts as atraumatically induced defect in the articular cartilage. This may occuras a fragmentation of the original tissue from the joint margins orother defect which compromises the surface and integrity of thearticular cartilage. Exposure of the supporting subchondral bone tosynovial fluid and the intermittent pressures of the synovial fluidgenerated by repeated joint movement (repeated stress and trauma oftraining or racing) can lead to progressive subchondral bone sclerosisand eventual dislodging of the chip or bone fragment. Left untreated,the resulting damage often becomes progressive and DJD results (see,e.g., Nixon et al., “Equine Fracture Repair,” W. B. Saunders Co., 1996(ISBN 0-7216-6754-6)).

Under either scenario, once compromised, the damage to articularcartilage is usually permanent. In general, once damaged, therapy isnormally directed at limiting or reducing joint inflammation, limitingthe release of inflammatory mediators, removal of the inciting cause(e.g., the chip) and replacement of synovial fluid components. Thesemeasures are combined with a period of rest to allow for healing andfibrocartilage deposition at the affected area. The long termtherapeutic objective is directed at slowing the progression ofdegenerative processes and controlling the clinical signs of DJD.Prevention is often aimed at limiting joint inflammation before damageto cartilage occurs and in providing proper nutritional support.

The treatment of connective tissue afflictions can be quite problematic.A simple decrease in the stress to which the connective tissue issubjected is often not an option, especially in the case of athletes andanimals such as race horses. Consequently, treatment is often directedat controlling the symptoms of the afflictions and not their causes,regardless of the stage of the degenerative process. Presently,steroids, such as corticosteroids and NSAIDs, are widely used for thetreatment of these ailments (Vidal, et al., Pharmocol. Res. Commun.,10:557-569 (1978)). However, drugs such as these, which inhibit thebody's own natural healing processes, may lead to further deteriorationof the connective tissue.

Connective tissue, for example articular cartilage, is naturallyequipped to attempt to repair itself by manufacturing and remodelingprodigious amounts of collagen and proteoglycans (PGs). This ongoingprocess is placed under stress when an injury occurs. In such cases, theproduction of connective tissue matrix (collagen and proteoglycans) candouble or triple over normal levels, thereby increasing the demand forthe building blocks of both collagens and proteoglycans. The buildingblocks for collagen are amino acids, especially proline, glycine andlysine. Proteoglycans are large and complex macromolecules comprisedmainly of long chains of modified sugars called glycosaminoglycans(GAGs) or mucopolysaccharides. The terms glycosaminoglycans andmucopolysaccharides are understood in the art to be interchangeable. Dueto their dense negative ion content, proteoglycans molecules are able toattract and retain water within the cartilage formation specifically forlubrication. Proteoglycans provide the unique mechanical properties forflexibility, resiliency, and resistance to and recovery undercompressive forces.

Glucosaminoglycans are polysaccharides which occur widely in the animalkingdom. Glucosaminoglycans that are present in the tissues ofvertebrate animals have mainly a linear structure which is repetition ofa disaccharide units composed of two monosaccharides. Five kinds ofglucosaminoglycans are found in the tissues and fluids of vertebrates:chondroitin sulfates, keratin sulfates, dermatan sulfates, heparinsulfates, and hyaluronic acid.

Proteoglycans and collagen are the chief structural elements of allconnective tissues. Their synthesis is essential for proper maintenanceand repair of connective tissues. In vitro, the introduction ofglucosamine, a key precusor for GAGs, has been demonstrated to increasethe synthesis of collagen and GAGs in fibroblasts. In vivo, topicalapplication of glucosamine has enhanced wound healing. Glucosamine hasalso exhibited reproducible improvement in symptoms and cartilageintegrity in humans with osteoarthritis (L. Bucci, NutritionalSupplement Advisor, July 1992)).

The major glycosaminoglycans found in cartilage are chondroitin sulfate,dermatan sulfate, keratan sulfate and hyaluronic acid (also known ashyaluronan or HA). Heparin sulfate is also a glycosaminoglycan, althoughit is not a component of articular cartilage. Newer names forproteoglycans sometime reference function of the core protein within themolecule found in chondroitin sulfate and keratin sulfate, e.g.,aggregan, a large proteoglycan aggregates with hyaluronin, or referencelocation (e.g., decorin (dermatan sulfate), which decorates type Icollagen fibrils), or reference primary structure, biglycan which hastwo glysoaminoglycan chains. Chondrocytes are active cells within thecartilage matrix, which manufacture new collagen and proteoglycanmolecules while excreting enzymes, which aid in removal of damagedcartilage and proteoglycans.

Chondroitin sulfate is broken down into sulfate disaccharides andN-acetyl galactosamine. Chondroitin sulfate, as CS4 and CS6 within thebody, is thought to be an essential glycosaminoglycan which binds waterto the articular cartilage matrix and is necessary for the formation ofproteoglycans.

In particular, chondroitin sulfate is a long hydrophilic chain ofrepeating sugars. This glycosaminoglycan binds to proteoglycan moleculesaiding in water and nutrient transportation within the articularcartilage. Chondroitin in its sulfate form includes galactosamine, aprimary substrate of hylauronan and a disaccharide pathway forproteoglycan synthesis secondary to the hexosamine pathways utilized forglycosaminoglycan production. Chondroitin sulfate chains comprise thespace formation of the cartilage matrix and integral parts of theproteoglycan molecule. Chondroitin stimulates the production ofproteoglycans, glycosaminoglycans, and collagen, which are the buildingblocks of healthy cartilage. Chondroitin sulfate also inhibits thesecretion of degenerative enzymes by the chondrocytes within articularcartilage. Chondroitin sulfates are non-toxic and work synergisticallywith glucosamine to hydrate and repair articular cartilage.

Hylauronan is an integral part of both synovial fluid and articularcartilage. Within the articular cartilage, hylauronan providesviscoelastic properties allowing ease of motion between opposingsurfaces and increasing compressive resistance. Within the synovium,hylauronan, as a component of synovial fluid, provides an effectivebarrier regulating the introduction of plasma components. Under normalconditions, the body will synthesize sufficient amounts of basecomponents to maintain and grow healthy articular cartilage, whilelimiting the production and release of destructive proteinases,inflammatory mediators and catabolic enzymes.

Glucosamine, as glucosamine 5-phosphate, is naturally occurring withinthe body and is a component in the biosynthesis of glycosaminoglycans,proteoglycans, hyaluronan, and collagen. Glucosamine is available inexogenous forms, glucosamine sulfate sodium, glucosamine hydrochlorideand N-acetyl D-glucosamine. N-acetyl D-glucosamine is also a derivativeof glucose obtained by chemical hydrolysis of chitin. Thispolysaccharide is readily soluble in water and extremely bioavailable.N-acetyl D-glucosamine binds to glucuronic acid as well as galactosemaking it a precursor to hyaluronic acid, keratan-sulfate andchondroitin sulfate. This unique derivative aids in proteoglycan,collagen and glycosaminoglycan production. N-acetyl D-glucosamine hasalso been shown to aid in the healing of soft tissue injury.D-Glucuronic acid is a key substrate comprising one half of thehyaluronan molecule, the other being N-acetyl D-glucosamine.

There have been countless therapeutic approaches for management of jointdisease, providing nutritional supplementation of metabolic precursorsto the diet to aid in the biosynthesis of proteoglycans, GAG's,hyaluronan, and collagen (see, U.S. Pat. Nos. 5,364,845 and 5,587,363).Numerous other disclosures also suggest the introduction of nutritionalsupplements as therapy for the treatment of connective tissues. Forinstance, U.S. Pat. No. 3,683,076 to Rovati et al. teaches thatglucosamine sulfates are useful to treat arthritic conditions. U.S. Pat.No. 3,697,652 to Rovati et al. discloses that N-acetyl glucosamine canbe used to treat degenerative afflictions of the joints. U.S. Pat. Nos.5,364,845, 5,587,363, 6,492,349, 6,271,213, and 6,583,123 to Hendersonet al. teach that glucosamine, chondroitin, manganese, and/orS-Adenosylmethionine (SAM) are used to protect and repair connectivetissue. U.S. Pat. No. 6,632,804 to Ekanauake teaches that ferrous ionand an ascorbate, and glucodamine derivative are useful in treatingosteoarthritis. U.S. Pat. No. 6,645,948 to Prtito et al. teaches anutritional composition for treating connective tissue including aglucosamine salt, chondroitin sulfate, collagen and sodium hyaluronate.

In U.S. Pat. No. 5,840,715 to Florio, N-acetyl glucosamine sulfate,chondroitin sulfate, gamma linolenic acid ercosapentaenoic acid anddocosahexaneoic acid, and manganese aspartate are combined to treatarthritis symptoms. U.S. Pat. No. 5,916,565 to Rose et al. teaches acomposition comprised of D-glucosamine hydrochloride, chondroitinsulfate, cayenne, ginger, turmeric, yucca, Devil's Claw, nettle leaf,Black Cohosh, alfalfa, and celery seeds to repair and maintain damagedtissues in joints of vertebrates. In U.S. Pat. No. 5,922,692, Marinodiscloses that glucosamine sulfate and chondroitin sulfate can be addedto foodstuffs. Additional related art discloses pharmaceuticalcompositions and methods for the treatment of connective tissue inhumans and animals, such as U.S. Pat. Nos. 4,216,204, 4,782,046,4,808,576, 4,837,024, 5,141,928, 5,840,715, 5,442,053, and 5,929,050.

Stem cells are cells found in most multi-cellular organisms. They arecapable of retaining the ability to reinvigorate themselves throughmitotic cell division and can differentiate into a diverse range ofspecialized cell types. The two broad types of mammalian stem cells are:embryonic stem cells that are found in blastocysts, and adult stem cellsthat are found in adult tissues. The two classical properties of stemcells are self-renewal and potency. Self-renewal refers to the abilityto go through numerous cycles of cell division while maintaining theundifferentiated state, and potency refers to the capacity todifferentiate into specialized cell types. Potency specifies thedifferentiation potential to differentiate into different cell types ofthe stem cells. For instance, totipotent stem cells are cells producedfrom the fusion of an egg and sperm cell, as well as the first fewdivisions of the fertilized egg, and they can differentiate intoembryonic and extra-embryonic cell types. Pluripotent stem cells are thedescendants of totipotent cells and can differentiate into cells derivedfrom any of the three germ layers. Multipotent stem cells can produceonly cells of a closely related family of cells (e.g. hematopoietic stemcells differentiate into red blood cells, white blood cells, platelets,etc.). Unipotent cells can produce only one cell type, but have theproperty of self-renewal which distinguishes them from non-stem cells(e.g. muscle stem cells). All such forms of stem cells can be consideredto be progenitor cells which are not terminally differentiated.

Progenitor cells refer to immature or partially undifferentiated cells,typically found in post-natal animals Like stem cells, progenitor cellshave a capacity for self-renewal and differentiation, although theseproperties may be limited depending upon the type of cell. Embryonicstem cells are true stem cells in that they are pluripotent and showunlimited capacity for self-renewal. In contrast, many cells termedadult stem cells are more commonly termed progenitor cells, as theircapacities for unlimited self renewal and plasticity have not beencomprehensively demonstrated. The majority of progenitor cells liedormant or possess little activity in the tissue in which they reside.They exhibit slow growth and their main role is to replace cells lost bynormal attrition. Upon tissue damage or injury, progenitor cells can beactivated by growth factors or cytokines, leading to increased celldivision important for the repair process. Examples of progenitor cellsinclude satellite cells found in muscle and the transit-amplifyingneural progenitors of the rostral migratory stream. Bone marrow stromalcells, basal cell of epidermis have 10% of progenitor stem cell,although they are often classed as stem cells due to their highplasticity and potentially unlimited capacity for self renewal.

Periosteum is a membrane that lines the outer surface of all bones,except at the joints of long bones. Endosteum lines the inner surface ofall bones. Periosteum consists of the irregular type of dense connectivetissue. Periosteum is divided into an outer “fibrous layer” and inner“cambium layer”. The fibrous layer contains fibroblasts while thecambium layer contains progenitor cells which develop into osteoblasts.These osteoblasts are responsible for increasing the width of a longbone, and the overall size of the other bone types. After a bonefracture, the progenitor cells develop into osteoblasts andchondroblasts which are essential to the healing process. As opposed toosseous tissue, periosteum has nociceptive nerve endings, making it verysensitive to manipulation. It also provides nourishment by providing theblood supply. Periosteum is attached to bone by strong collagenousfibers called Sharpey's fibres, which extend to the outercircumferential and interstitial lamellae. It also provides anattachment for muscles and tendons.

Mesenchymal stem cells or Marrow Stromal Cells (MSCs) are multipotentstem cells that can differentiate into a variety of cell types, excepthematopoietic cells. Cell types that MSCs have been shown todifferentiate into in vitro or in vivo include osteoblasts,chondrocytes, myocytes, adipocytes, and beta-pancreatic islets cells.Like the connective tissue cells, Stromal cells, MSCs also provide thesupportive structure in which the functional cells of the tissue reside.In addition, MSCs play roles in repair of tissue. Because MSCs canencompass multipotent cells derived from other non-marrow tissues, suchas adult muscle side-population cells or the Wharton's jelly present inthe umbilical cord, as well as in the dental pulp of deciduous babyteeth, yet do not have the capacity to reconstitute an entire organ,MSCs may also stand for Multipotent Stromal Cells.

Because in adult organisms, stem cells and progenitor cells act as arepair system for the body, replenishing specialized cells, but alsomaintain the normal turnover of regenerative organs, these cells havebeen utilized for treating skeletal and other connective tissuedisorders. For instance, U.S. Pat. Nos. 5,226,914; 5,197,985; 5,735,542;and 6,010,696 to Caplan et al. provide methods and devices for treatingand repairing connective tissue damage by enhancing bone marrow orhematopoietic progenitor cell engraftment and implantation anddifferentiation of marrow-derived mesenchymal cells by applyingculturally expanded purified marrow-derived mesenchymal cells to an areaof connective tissue damage under conditions suitable fordifferentiating the cells into the type of connective tissue necessaryfor repair, and methods and devices for using the purifiedmarrow-derived mesenchymal cells in order to enhance the production ofhematopoitic cells.

Mesenchymal stem cells are the formative pluripotent blast cells foundin the bone that are capable of differentiating into any of the specifictypes of connective tissues (i.e., the tissues of the adipose, areolar,osseous, cartilaginous, elastic, and fibrous connective tissues)depending upon various environmental influences. Although these cellsare normally present at very low frequencies in bone marrow and othermesenchymal tissues, a process for isolating, purifying, and greatlyreplicating the mesenchymal stem cells in culture is described in U.S.Pat. No. 5,486,359. Under selected conditions, the mesenchymal stemcells can be induced to differentiate into different types of skeletaland connective tissues such as bone, cartilage, tendon, ligament,muscle, other connective tissues and marrow stroma.

Moreover, U.S. Pat. No. 5,842,477 provides methods of making and/orrepairing cartilage in vivo by implanting into a patient, at a site ofcartilage damage or loss, a biocompatible, non-living three-dimensionalscaffold or framework structure in combination withperiostel/perichondrial tissue, and administering a preparation ofchondrocytes and/or other stromal cells, such as chondrocyte progenitorcells, to the site of the implant before, during or after implantationof the scaffold and/or the periosteal/perichondrial tissue. It isprovided that the periosteal/perichondrial tissue can be used to holdthe scaffold in place at the site of implantation and also provides asource of stromal cells, e.g., chondrocytes and/or chondrocyteprogenitor cells, for attachment to the scaffold in vivo, and the seededstromal cells provides not only a readily-accessible source ofchondrocytes and/or other stromal cells for attachment to the scaffoldbut also provides a rapid and efficient means of inducing chondrogenesisas well as migration of stromal cells from the surrounding in vivoenvironment to the scaffold via factors produced by the stromal cells ofthe preparation. Additionally, it is provided that the seeded stromalcells can be genetically engineered to express gene products beneficialto growth, implantation and/or amelioration of disease conditions,resulting in the efficient production of new cartilage in vivo, that isuseful in the production/repair of articular cartilage in patientssuffering from degenerative connective tissue diseases such asrheumatoid and/or osteoarthritis as well as in patients who havecartilage defects due to trauma.

Furthermore, U.S. Pat. No. 6,936,281 to Seshi provides isolatedpluri-differentiated human mesenchymal progenitor cells (MPCs) fromDexter-type cultures, and method of isolating and using these cells fordiagnostic uses, and for therapeutic uses to enhance the engraftment ofhematopoietic progenitor cells, enhance bone marrow transplantation, oraid in the treatment or prevention of graft-versus-host diseases (GvHD).

While all the above references have been described as being effectivefor their intended use, there remains a need in the art for atherapeutic composition which demonstrates enhanced effectiveness in thetreatment of connective tissues, exhibits other improved beneficialproperties, and provides even wider applications. The present inventionmeets these needs at least in part.

SUMMARY OF THE INVENTION

The present invention provides an enhanced effectiveness in thepreventing and treating connective tissue damage, or specifically in therepair of cartilage in an affected site. In one embodiment, the presentinvention provides a pharmaceutical preparation, and method of usethereof, for treating connective tissue damage and for repair cartilagein an affected site in man and in animals. The pharmaceuticalpreparation described herein comprises a glycosaminoglycan compositionin combination with stem cells. Methods of treatment are provided hereinusing the glycosaminoglycan composition in combination with stem cells,either as a pharmaceutically acceptable admixture or in concurrent orsequential administration. The glycosaminoglycan compositions havefurther utility in combination with stem cells as a culture media and asa cryoperservative.

As used herein, the glycosaminoglycan composition described hereincomprises one or more glucosaminoglycans (GAGs) and is formulated intoany pharmaceutically acceptable formulations, including, but not limitedto, a sterile solution, suspension or gel for direct application orintra-articular, intramuscular, intravenous, or other parenteral orsystemic administration. Any GAGs can be included in theglycosaminoglycan composition described herein. In certain embodiments,the glycosaminoglycan composition comprises, or consists essentially of,chondroitin sulfate, glucosamine, and hyaluronan. In certainembodiments, the glycosaminoglycan composition comprises, or consistsessentially of, glucosamine and hyaluronan.

In one embodiment, the glycosaminoglycan composition comprises a mixtureof chondroitin sulfate, glucosamine and hyaluronan, and can be stored ina single container at room temperature, in a refrigerator or a freezer.In yet another embodiment, the glucosamine, such as N-acetylD-glucosamine, is stored in a separate container at room temperature, ina refrigerator or freezer, and can be mixed with the chondroitin sulfateand hyaluronan mixture before administration. In yet another embodiment,the glycosaminoglycan composition comprises chondroitin sulfate,hyaluronan, and glucosamine, such as N-acetyl D-glucosamine, all mixedtogether and stored in a single container ready for administration. Inyet another embodiment, the glycosaminoglycan composition is thePOLYGLYCAN® composition (ArthroDynamic Technologies, Lexington, Ky.)consisting essentially of a therapeutically effective amount ofchondroitin sulfate, N-acetyl D-glucosamine, and hyaluronan (hyaluronicacid) which is combined with a stem cell preparation for culturing orcryopreserving the stem cells, or for therapeutically preventing ortreating connective tissue damage.

In the joint, for example, chondroitin sulfate acts to stimulate theproduction of proteoglycans, glycosaminoglycans, and collagen, inhibitsdegenerative enzymes excreted by the chondrocytes, and synoviocytes, andaids in nutrient transportation within the synovial fluid. Glucosamine,in particular N-acetyl D-glucosamine, increases the synoviocyte andchondrocyte production and subsequent availability of endogenoushyaluronan by the direct in situ inclusion of its prime substratesgalactosamine (through chondroitin sulfate assimilation) and N-acetylD-glucosamine. The exogenous hyaluronan acts to replace depletedendogenous hyaluronan and to lubricate and coat healthy as well asdamaged articular tissue during the reparative process. The above modesof action are believed accurate; however, the claimed uses of thepresent compositions are not limited to such hypothesized mechanisms ofactivity for achieving efficacy.

In one embodiment, the chondroitin sulfate in the composition ischondroitin 4-sulfate (CS4), chondroitin 6-sulfate (CS6), or a mixtureof both CS4 and CS6. A therapeutically effective amount of chondroitinsulfate and N-acetyl D-glucosamine can be from between about 0.5 gramsto about 1.5 grams of per unit dose, respectively, and thetherapeutically effective amount of hyaluronan can be from about 10 mgto about 50 mg per unit dose.

In yet other embodiments, the stem cells are cultured or frozen with oneor more appropriate cryoprotectants including the glycosaminoglycancompositions described herein. As used herein, the term “stem cells”refers to cells which are not terminally differentiated, including cellscapable of retaining the ability to be passaged through mitotic celldivision, and ultimately differentiating into a diverse range ofspecialized cell types, including, but not limited to, loose, denseregular, and elastic connective tissue cell types. In one embodiment,the stem cells described herein are presursors to mesenchyme and/orstromal cells including, but not limited to, osteoblasts, chondrocytes,chondrocyte progenitor cells including mesenchymal stem cells or MSCs,fibroblasts, fibroblast-like cells, and other stromal cells capable ofproducing collagen types and proteoglycans which are typically producedin cartilaginous tissues. In yet another embodiment, the stem cellsdescribed herein are stromal cells capable of producing adipose oradipose-like cells for adipose. In yet another embodiment, the stemcells described herein are mesenchyme that differentiates intohematopoietic tissue.

In yet another embodiment, the stem cells are chondroigenic stem and/orprogenitor cells including mesenchymal stem cells or MCSs. In furtherembodiments, the mesenchymal stem cells or MCSs are human mesenchymalstem cells isolated from a human tissue specimen. In yet otherembodiments, the mesenchymal stem cells or MCSs are animal mesenchymalstem cells isolated from an animal tissue specimen. In yet otherembodiments, the chondroigenic stem and/or progenitor cells can beobtained from the patient (or animal subject) or a histocompatibledonor. The chondrocytes, progenitor cells, fibroblast-like cells andother cells and/or elements that comprise the stroma may be fetal oradult in origin, and may be derived from convenient sources such asadipose, cartilage, bone, skin, ligaments, tendons, muscles, placenta,umbilical cord, etc. For example, stromal cells such as chondrocytes maybe derived from any type of cartilage, including but not limited to,hyaline cartilage, costal cartilage, fibrous cartilage, etc., which canbe obtained by biopsy (where appropriate) or upon autopsy.

Chondrocyte progenitor cells may be derived from various sourcesincluding bone marrow, periosteum, perichondrium or various sources ofundifferentiated human mesenchyme. Fibroblasts can be obtained inquantity rather conveniently from foreskin, preferably fetal foreskin,or, alternatively, any appropriate cadaver organ. Fetal cells, includingfibroblast-like cells and chondrocyte progenitor cells, may be obtainedfrom umbilical cord or placenta tissue or umbilical cord blood. Stemcells from a variety of sources may be used in the present invention,including stromal cells derived from cells of fetal origin which may beviewed as “universal donors” so as to minimize the risk of immunologicalrejection, or adult origin, particularly in the case of autologous cellisolation and transplantation.

The stem cells used in the present invention may be readily isolated bydisaggregating an appropriate tissue and/or embryo which is to serve asthe source of the cells. Once the tissue and/or embryo has been reducedto a suspension of individual cells, the suspension can be fractionatedinto subpopulations from which precursors of chondrocytes, fibroblastsand/or other stromal cells can be obtained. Furthermore, oncechondrocytes or chondrocytes progenitor cells have been isolated, theirpopulation can be expanded mitotically in vitro in order to obtain thecell preparation for the combination with the other glucosaminoglycansin the composition disclosed in the present invention. The stem cellsused in the present invention may be isolated and/or purified from thecultured primary and/or transferred or “passaged” cells. Moreover, oncethe stem cells of the present invention have been established inculture, they may be maintained or stored in cell “banks” comprisingeither continuous in vitro cultures of cells requiring regular transfer,or cells which have been cryopreserved. In one embodiment, the embryosand the stem cells provided herein are cultured in the glycosaminoglycancompositions described herein, and/or frozen in the presence of one ormore suitable cryoprotectant, including the glycosaminoglycancompositions described herein, so that the stem cells provided hereinare properly protected during the cryopreservation process, yieldingimproved viability. The cryoprotected stem cells constitute a bank ofcells, portions of which can be “withdrawn” by thawing and then used forpreparing the pharmaceutical preparation described herein.

The stem cells used in the present invention may be geneticallyengineered to produce gene products that promote the successfulproduction or repair of cartilage or other connective tissue at a defectsite or for use in gene therapies. The stem cells used in the presentinvention may also be genetically engineered to express one or moregenes that would exert a therapeutical effect, or replace deficientgenes in a patient or animal for gene replacement therapy. Furthermore,the stem cells used in the present invention may also be geneticallyengineered to knock out expression of factors that promote inflammation,rejection, or other undesired side effects at a defect site.

The present invention further provides that the stem cells used in thepresent invention further comprise other cells aid in the production ofconnective tissues and/or cartilage. For example, other cells found inloose connective tissue may be included along with chondrocytes orfibroblasts. Such cells include, but are not limited to, endothelialcells, pericytes, macrophages, monocytes, plasma cells, mast cells, andadipocytes. As used herein, the connective tissue types include, but arenot limited to, bone, cartilage, tendon, ligament, dermis, periosteum,perichondrium, skin, or adipose.

As used herein, the pharmaceutical preparation of the present inventionis formulated in a suitable form, including, but not limited to, a formof sterile solution, suspension, or a gel or paste-like formulation. Inone embodiment, the glycosaminoglycan composition described herein canbe applied directly (e.g., direct injection) into the affectedconnective tissue, e.g., a joint, a tendon, a ligament or bone, followedby subsequent direct application of the stem cells or the pharmaceuticalacceptable formulations comprising the stem cells. In yet otherembodiments, the pharmaceutical preparation of the present invention maybe formulated for intra-articular and/or systemic or parenteraladministration, in which the glycosaminoglycan composition and the stemcells or a pharmaceutical acceptable formulation comprising the stemcells may be administered either separately, or mixed togetherimmediately before administration. Systemic administrations can include,but are not limited to, intramuscular, intravenous or subcutaneousinjection.

In yet another embodiment, each of the pharmaceutical preparation, theglycosaminoglycan composition, the stem cells, or the formulationcomprising the stem cells of the present invention can be attached to asheet of material adapted for implantation onto or between tissues of amammalian body. Each of the pharmaceutical preparation, theglycosaminoglycan composition, the stem cells, or the pharmaceuticallyacceptable formulation comprising the stem cells can be impregnated intoa porous gauze-like material or coated onto a gauze-like material orjoined to the material by adhesion and/or capillary action. The materialmay be either a permanent implant or it may be biodegradable. In yetanother embodiment, each of the pharmaceutical preparation, theglycosaminoglycan composition, the stem cells, or the pharmaceuticalacceptable formulation comprising the stem cells is attached to abandage or other surgical materials, including, but not limited to,surgical suture material, surgical staple, or a device such as a buckle.

The pharmaceutical preparation described herein further may optionallycomprise one or more other therapeutic agents, including, but notlimited to, synthetic and non-synthetic corticosteroid agents,nonsteroidal anti-inflammatory drugs, analgesics, antirheumatics,immunoregulators, immunosuppressant, articular function augmenters,interleukin production inhibitors, or grow factor, all of which havetherapeutic effects. Any drugs, agents, compounds, known and/or to bedeveloped, showing any desired therapeutic effects are within the scopeof this invention. In other embodiments, the invention may specificallyexclude one or more of the above therapeutic agents. In yet anotherembodiment, the stem cells, or the pharmaceutically acceptableformulation comprising the stem cells, may further comprise other cellsthat aid in the production of one or more connective tissue. These othercells include, but are not limited to, endothelial cells, pericytes,macrophages, monocytes, plasma cells, mast cells, or adipocytes.

The present invention provides that the pharmaceutical preparationdescribed herein enhances effectiveness promoting healthy growth ofconnective tissue and in the treatment and repair of connective tissuedamage. As used herein, the term “connective tissue” refers to loose,dense regular, and elastic connective tissues. The loose connectivetissues comprises constituent fibers including, but not limited to,collagenous fibers, elastic fibers, reticular fibers. The looseconnective tissue also refers to an adipose tissue. The dense regularconnective tissues include, but are not limited to, tendons, ligaments,cartilage, skeleton, and other fibrous connective tissues. Theconnective tissues used herein also refer to blood.

The present invention further provides that the pharmaceuticalpreparation described herein can be used in the prevention, treatment,and repair of connective tissue damage, which includes any primary orsecondary diseases or injuries to the connective tissues in humans oranimals. Such diseases or injuries include, but are not limited to,arthritic diseases, osteoarthritis (OA), rheumatoid arthritis (RA),osteochondrosis dessicans (OCD), cartilage damage, joint injuries, jointinflammation, joint synovitis, degenerative joint disease (DJD), postsurgical DJD, traumatic injuries, fractures, tendon damage, ligamentdamage, skeletal damage, musculoskeletal damage, bone damage, fiberdamage, adipose tissue damage, blood cell damage, and plasma damage.

The present invention further provides a method for preventing,treating, and repairing connective tissue damage in humans or in animalscomprising administering to a man or animal in need thereof, apharmaceutical preparation of the present invention. In one embodiment,the pharmaceutical preparation described herein comprises aglycosaminoglycan composition comprising, or consisting essentially of,a therapeutically effective amount of chondroitin sulfate, glucosamine,e.g., N-acetyl D-glucosamine, and hyaluronan, in combination with stemcells and/or a pharmaceutically acceptable formulation comprising thestem cells. In yet another embodiment, the pharmaceutical preparationdescribed herein comprises POLYGLYCAN® composition that consistsessentially of chondroitin sulfate, N-acetyl D-glucosamine, andhyaluronan, in combination with stem cells and/or a pharmaceuticallyacceptable formulation comprising the stem cells. In one embodiment, thestem cells described herein are progenitor mesenchymal or stromal cellsincluding, but not limited to, osteoblasts, chondrocytes, includingmesenchymal stem cells or MSCs, fibroblasts, fibroblast-like cells, andother stromal cells capable of producing collagen types andproteoglycans which are typically produced in cartilaginous tissues. Inyet another embodiment, the stem cells described herein are stromalcells capable of producing adipose or adipose-like cells for adipose. Inyet another embodiment, the stem cells described herein are mesenchymalthat differentiate into hematopoietic tissue.

In one embodiment, the present invention provides that thepharmaceutical preparation described herein comprises a mixture of theglycosaminoglycan composition and the stem cells and/or the stem cellformulation in a suitable container for a period of time withoutdegradation of each ingredient in the proteoglycan position and the stemcells. In one embodiment, the present invention provides that thepharmaceutical preparation described herein comprises theglycosaminoglycan composition and the stem cells or the stem cellformulation comprising the stem cells, in which the glycosaminoglycancomposition and the stem cells or the stem cell formulation are mixedimmediately before administering to the patient (human or animalsubject) in need. In alternative embodiments, the stem cells or the stemcell formulation described herein can be administered before, during orafter the administration of the glycosaminoglycan composition describedherein.

DETAILED DESCRIPTION OF THE INVENTION

Additional objects, advantages and other novel features of the inventionwill be set forth in part in the description that follows and in partwill become apparent to those skilled in the art upon examination of theforegoing or may be learned with the practice of the invention.Additionally, throughout this document, various publications and patentshave been cited, the contents of which are incorporated herein byreference in their entirety.

Set forth in greater detail below are specific details related to apharmaceutical preparation which demonstrates enhanced effectiveness inthe treatment of connective tissue damage. The pharmaceuticalpreparation provided herein comprises a glycosaminoglycan compositioncomprising a therapeutically effective amount of chondroitin sulfate, asuitable glucosamine derivative, e.g., N-acetyl D-glucosamine, and asuitable hyaluronan (hyaluronic acid), in combination with stem cells ora pharmaceutically acceptable formulation comprising the stem cells.

Preparation of the Glycosaminoglycan Composition

The present invention provides that the pharmaceutical preparationdescribed herein comprises a glycosaminoglycan composition. As usedherein, the term “glycosaminoglycan composition” refers to anypharmaceutically acceptable composition comprising one or moreglucosaminoglycans (GAGs), or pharmaceutically acceptable salts thereof,with or without a pharmaceutically acceptable carrier. Theglycosaminoglycans can be selected from the group selected from, orconsisting essentially of, chondroitin sulfate and hyaluronan. In oneembodiment, the glycosaminoglycan composition described herein compriseschondroitin sulfate and hyaluronan, and optionally N-acetylD-glucosamine. In one embodiment, the glycosaminoglycan compositiondescribed herein comprises hyaluronan and N-acetyl D-glucosamine, andoptionally chondroitin sulfate. In yet another embodiment, theglycosaminoglycan composition described herein is a POLYGLYCAN®composition consisting essentially of chondroitin sulfate, N-acetylD-glucosamine, and hyaluronan, wherein by combining the three componentsdescribed herein, particularly in combination with a cellular therapy,the composition provides a synergistic effect in the treating connectivetissue damage and repairing defective cartilage. Glycosaminoglycancompositions such as described in U.S. Pat. Nos. 6,979,679 and 7,485,629are hereby incorporated by reference in their entireties.

It is believed that chondroitin sulfate acts to stimulate the productionof proteoglycans, glycosaminoglycans, and collagen, inhibitsdegenerative enzymes excreted by the chondrocytes, and synoviocytes, andaids in nutrient transportation within the synovial fluid. It isbelieved that glucosamine derivatives, particularly N-acetylD-glucosamine, increase the synoviocyte and chondrocyte production andsubsequent availability of endogenous hyaluronan by the direct in situinclusion of its prime substrates galactosamine (through chondroitinsulfate assimilation) and N-acetyl D-glucosamine. It is further believedthat the exogenous hyaluronan acts to replace depleted endogenous HA andto lubricate and coat healthy as well as damaged articular tissue duringthe reparative process. The examples and proposed mechanisms of actionset forth herein are in no way intended to limit the scope of theinvention. Those of skill in the art will realize that, given theteachings provided herein, many variations of the compositions, methodsof use thereof, are possible that will fall within the scope of theinvention.

The glycosaminoglycan composition described herein provides a uniquemixture comprised of the isolated naturally occurring agents:chondroitin 4-sulfate (CS4) and chondroitin 6-sulfate (CS6), hyaluronanand a suitable glucosamine derivative, e.g., N-acetyl D-glucosamine Itcan be appreciated that, depending upon the target connective tissue,the suitable glucosamine derivative can be selected from any of theglucosamine derivatives including, but not limited to, glucosamine5-phosphate, glucosamine sulfate sodium, glucosamine hydrochloride,N-acetyl D-glucosamine, and mixtures thereof. Synthetic derivatives ofany of the identified naturally occurring agents can also be used in theinvention.

Chondroitin sulfates are one of the important components of theglycosaminoglycan composition described herein. In general, chondroitinsulfates are widely found in the connective tissues of animals in twoforms of repeating disaccharides of D-glucoronic acid and N-acetylgalactosamine: CS4 sulfate where n-acetyl galactosamine holds an estersulfate in its CS4 position or CS6 sulfate where the ester sulfate is inthe CS6 position. Both CS4 and CS6 chondroitin sulfate function in thearticular matrix as a major constituent. Chondroitin sulfates contributeto keep the cartilage matrix's normal characteristics through theincrease of the glucosaminoglycan pool used by the chondrocytes forproteoglycan synthesis, as well as slowing down the inflammatory processacting directly on the enzymes inhibiting the compliment cascade and byexhibiting anti-prostoglandin activity.

Another important component in the glycosaminoglycan compositiondescribed herein is hyaluronan and its salts (e.g., sodium hyaluronate).Hyaluronan is a natural constituent of connective tissues and synovialfluid composed of repeating disaccharide units each consisting ofD-glucoranic acid and N-acetyl D-glucosamine. Within the joint capsule,the surface of articular cartilage is covered by a thin layer of sodiumhyaluronate. It specifically interacts with cartilage proteoglycans toform a stabile aggregate. Within the synovial fluid it confers viscalelasticity as well as lubricating properties. Hyaluronan aids inproviding nourishment and waste removal from the articular matrix. Italso provides biochemical activity to help prevent excess fibrous tissuefrom forming in the cartilage matrix.

In addition, N-acetyl D-glucosamine is also one of the importantcomponents in the glycosaminoglycan composition described herein. TheN-acetyl D-glucosamine is a key compound for cartilage matrix synthesisas it enhances chondrocyte synthesis of glucosaminoglycans. N-acetylD-glucosamine also possesses the ability to enhance synthesis of keycomponents of synovial fluid by feeding both reactions necessary for theproduction of hyaluronan as well as for proteoglycans. Therefore, byreplacing specific glucosaminoglycans lost by the invasion of thediarthrodial joint during surgery and also providing the key moleculesto enhance and promote the restoration of normal hyaluronan andproteoglycan synthesis, the physician or veterinarian can be assured ofthe composition's capability to protect the joint as well as to aid inthe healing process.

As used herein, the chondroitin sulfate in the glycosaminoglycancomposition can be CS4, CS6, or a mixture of both CS4 and CS6. Thetherapeutically effective amount of chondroitin sulfate and N-acetylD-glucosamine can be from between about 0.5 grams to about 1.5 grams ofper unit dose, respectively, and the therapeutically effective amount ofhyaluronan can be from about 10 mg to about 50 mg per unit dose. In oneembodiment, the therapeutically effective amount comprises about 1 gramof CS4 chondroitin sulfate, or about 1 gram of CS6 chondroitin sulfateor about 1 gram of a mixture of CS4 and CS6 chondroitin sulfate per unitdose. In another embodiment, the therapeutically effective amount ofchondroitin sulfate is about 1 gram of chondroitin sulfate comprised ofabout 40% CS4 chondroitin sulfate and about 60% CS6 chondroitin sulfate.

A therapeutic amount of N-acetyl D-glucosamine can be about 1 gram ofN-acetyl D-glucosamine per unit dose of the composition. Therapeuticamounts of hyaluronan include from about 10 mg to about 50 mg ofhyaluronan per unit dose of the composition. Therapeutic amounts ofhyaluronan can be from about 20 to about 40 mg of hyaluronan per unitdose of the composition.

It can be appreciated by one of skill in the art that the hyaluronan canbe selected from among any of a number of commercially availablesources, such as commercially available sodium hyaluronate, and caninclude alternative salts and metabolic precursors or metabolitesthereof. Likewise there are numerous commercially available sources ofN-acetyl D-glucosamine and chondroitin sulfate and various alternativesalts or metabolic precursors or metabolites thereof that are availablefor use in the glycosaminoglycan composition described herein.

Another embodiment of the glycosaminoglycan composition provided hereincomprises a sterile solution or suspension comprising about 1 gram ofchondroitin sulfate as a mixture of about 40% CS4 and 60% CS6chondroitin sulfate; about 1 gram of N-acetyl D-glucosamine; and about20-40 mg but especially about 30 mg of hyaluronan (e.g., sodiumhyaluronate) per unit dose of the glycosaminoglycan compositiondescribed herein.

One example of a glycosaminoglycan composition provided herein comprisesa 10 ml unit dose, and is made as follows: one gram of chondroitinsulfate powder is admixed with one gram of N-acetyl D-glucosaminepowder. These powders are weighed, admixed and 2 ml of a 10 mg/mlsolution of sodium hyaluronate is added to the powder mixture. Theresultant mixture of chondroitin sulfate, N-acetyl D-glucosamine andsodium hyaluronate can be qs with approximately 10 ml of bacteriostaticwater to achieve a final volume of 10 ml. The final concentration ofchondroitin sulfate in the glycosaminoglycan composition describedherein can be 0.1 gram/ml or 10%. The final concentration of N-acetylD-glucosamine in the glycosaminoglycan composition described herein canbe 0.1 gram/ml or 10%, and the final concentration of sodium hyaluronatein the glycosaminoglycan composition described herein can be 0.2 ml/mlor 20%.

One embodiment of the glycosaminoglycan composition provided herein is,for example, POLYGLYCAN® that consists essentially of therapeuticamounts of chondroitin sulfate, N-acetyl D-glucosamine, and hyaluronan.The glycosaminoglycan composition described herein may specificallyexclude other therapeutic agents, such as analgesics, or mayspecifically include other therapeutic agents, such asimmunosuppressants or bactericides. The glycosaminoglycan compositiondescribed herein may specifically exclude chondroitin sulfate, andcomprise or consist essentially of N-acetyl D-glucosamine andhyaluronan.

In one embodiment, the glycosaminoglycan composition provided hereincomprises therapeutically effective amounts of chondroitin sulfate,N-acetyl D-glucosamine, and hyaluronan, wherein the molecular weight pereach supramolecule per unit dose of the glycosaminoglycan compositiondescribed herein is from between about 450,000 Daltons to about1,600,000 Daltons, preferably is from between about 500,000 Daltons toabout 1,400,000 Daltons, more preferably is from between about 550,000Daltons to about 700,000 Daltons, and most preferably is about 600,000Daltons. In yet another embodiment, the glycosaminoglycan compositionprovided herein comprises therapeutic amounts of chondroitin sulfate,N-acetyl D-glucosamine, and hyaluronan, wherein the molecular weight pereach supramolecule per unit dose of the glycosaminoglycan compositiondescribed herein is greater than about 450,000 Daltons, preferably isgreater than about 550,000 Daltons.

Preparation of the glycosaminoglycan composition provided herein may bemade by conventional methods. For example, to prepare theglycosaminoglycan composition of the invention, the above-describedingredients are combined as the active ingredient in intimate admixturewith or without a suitable carrier according to conventional compoundingtechniques. This carrier may take a wide variety of forms depending uponthe form of preparation desired for administration, e.g., oral,sublingual, nasal, guttural, rectal, transdermal, or parenteral.

The Stem Cell Preparation

The term “stem cell” as used herein refers to either (1) a multipotent,or lineage-uncommitted progenitor cell, which is isolated fromembryonic, fetal or adult tissues, such as adipose, bone marrow, blood,dermis and periosteum, that are naturally or induced to be capable ofdifferentiating into multiple specific types of mesenchymal orconnective tissues (i.e., the tissues of the body that support thespecialized elements; particularly adipose, osseous, cartilaginous,elastic, and fibrous connective tissues) depending upon variousinfluences from bioactive factors, such as cytokines, or (2) alineage-committed progenitor cell produced from the mitotic division ofa stem cell which will eventually differentiate into a chondrocyte.Unlike the stem cell from which it is derived, the lineage-committedprogenitor is generally considered to be incapable of an unlimitednumber of mitotic divisions and will eventually differentiate into achondrocyte. Therefore, a “stem cell” as used herein is selected fromany number of non-terminally differentiated cells.

Methods for isolating, purifying, and replicating these stem cells inculture, i.e. in vitro, for the stem cell preparations provided hereinare provided based on the conventional methods known in the art. Forexample, U.S. Pat. Nos. 5,197,985; 5,486,359; 5,226,914 to Caplan et al.(the entire contents of which are incorporated herein by reference)relate to culturing marrow-derived mesenchymal stem cells in vitro inthe presence of growth factors, applying these cells to a carrier topromote cell morphology, and implanting the carrier containing the cellsinto damaged articular cartilage. U.S. Pat. No. 6,936,281 to Seshi (theentire contents of which is incorporated herein by reference) providesmethods for isolating and purifying human mesenchymal progenitor cells(MPCs) from Dexter-type cultures, and their therapeutic uses fordiagnostic purposes, and to enhance the negraftment of hematopoieticprogenitor cells, enhance bone marrow transplantation, or aid in thetreatment or prevention of graft versus host disease.

A stem cell, or chondrocyte progenitor cell, of the invention can alsobe isolated from other tissue sources including adipose, such asfalciform tissue. Several exemplary commercially available systems existfor the isolation of such stem cells from adipose, including those fromInGeneron, Inc. (Houston, Tex.), MediVet America Inc., (Nicholasville,Ky.) and Vet-Stem, Inc. (Poway, Calif.). For example, stem cells of thepresent invention can be prepared using the Autologous Regenerative CellSystem (ARC System) provided by InGeneron, Inc., which is described indetail in the U.S. Patent Application Publication No. 2010/0285588 (theentire contents of which are incorporated herein by reference). The ARCsystem is designed for rapid point-of-care preparation of regenerativecells from adipose tissue in settings where small quantities of tissueare available, such as applications in veterinary medicine. The ARCsystem comprises an apparatus for isolating cells from adipose tissuethat includes a lipid separating apparatus having one or more dispersingports equipped with a plurality of pores (or other means of dispersion)and a cell separation assembly that includes a plurality of optionallyremovable filters of variable pore size. The cell separation assembly isarranged to interface with a lipoaspiration device and to selectivelyseparate and elute cells.

The ARC System comprises a unitary disposable apparatus and itsassociated components and instructions packaged in a kit, used forrapidly isolating and/or recovering stromal vascular fraction (SVF)cells and/or a reparative cell population comprising viablenon-adipocyte cells (e.g., preadipocytes, mesenchymal stromal cells,endothelial cells, endothelial progenitor cells, fibroblasts,macrophages and lymphocytes) from adipose tissue. The apparatus includesa container/digestion chamber adapted for processing adipose tissue intoindividual cells and small clusters of cells to a diameter of about 1000microns or less and a lipid separating unit adapted to separate theprocessed adipose tissue without the need for centrifugation into alipid layer enriched in adipocytes and an aqueous layer enriched instromal vascular fraction cells. Stromal cells isolated from adiposetissue can yield cell preparations useful for the repair of articularcartilage. Additionally, these stromal cells can be cultured todifferentiate into cells having neuronal characteristics. Likewise,adipose-derived stromal cells can be used as a source in generatinghematopoietic cells, osteogenic cells, endothelial cells, adipocytes andmyocytes of skeletal and smooth muscle.

More specifically, the unitary disposable apparatus provided forrecovering cells from a biological tissue without a need forcentrifugation includes a digestion chamber adapted to receive anddissociate the biological tissue into a digestion mixture includingcells and cell clusters and a lipid separating unit in fluidcommunication with the digestion chamber and adapted for phaseseparation of the digestion mixture into an aqueous cellular layer and alipid layer that includes free lipids and cells that contain lipiddroplets such as adipocytes. The aqueous cellular layer refers to alayer of cells that lack internal lipid droplets as are found inadipocytes. The digestion chamber of the apparatus is divided by aninternal digestion mesh into post-digestion chamber and a predigestionchamber. The digestion chamber and associated fluid conduits are adaptedto provide a recirculating fluid path through the pre-digestion andpost-digestion chambers via a recirculating tubing circuit. Typically, amotive force for a fluid flow through the apparatus is provided by oneor more pumps.

A heat exchanger is disposed in functional communication with therecirculating tubing circuit and the fluid flow is directed through theheat exchanger by the motive force of the one or more pumps. The heatexchanger is able to adapt the temperature of fluid flowing through thedigestion chamber and thereby control activity of digestion enzymesutilized to digest the connective tissues that hold the tissue together.The one or more pumps and the heating exchanger are external to theapparatus and the apparatus is a one-time use disposable unit.

The internal digestion mesh is fixed in a vertical cylindricalorientation within the digestion chamber such that the pre-digestionchamber and the post-digestion chamber are in a concentric orientation.In one configuration, the outer concentric chamber is the predigestionchamber and the inner chamber is the post digestion chamber. In thisconfiguration, a maximum surface area and exposure to fluid flow isprovided to the pre-digested tissue and clogging of the digestion meshis minimized At least one dispersing filter is disposed in the fluidcommunication between the digestion chamber and the lipid separatingunit. The dispersing filter can also be disposed in a fixed horizontalplane and is arranged in a perpendicular orientation to the digestionmesh.

In one aspect, the apparatus is a unitary combination of digestionchamber, dispersing filter and lipid separating unit in a singlesterilizable and disposable unit that forms closed flow path from theinitial introduction of tissue into the unit until isolated individualcells and small clusters of cells are removed from the unit. In suchapparatus, the bottom of the digestion chamber forms a top of the lipidseparating unit and the dispersing filter is disposed circumferentially,and integrated near a top aspect, of the lipid separating unit such thatthe surface area of the dispersing filter is maximized and the size ofthe lipid separating unit is minimized

The digestion chamber of the apparatus can also be divided by aninternal digestion mesh into a post-digestion chamber and a concentricpre-digestion chamber. In this configuration, the digestion chamber as awhole includes at least one inlet port configured to introduce adiposecontaining tissue into the pre-digestion chamber and at least one outletport configured to recover a digested cell mixture out of thepost-digestion chamber and a fluid conduit connecting the pre-digestionchamber and the post-digestion chamber and adapted to provide arecirculating fluid flow from the post-digestion chamber and back to thepre-digestion chamber. The post digestion chamber is in fluidcommunication with a lipid separating unit and the lipid separating unithas at least one dispersing port equipped with a plurality of dispersingpores through which the digested cell mixture enters the lipidseparating unit. The pores of the dispersing port effect furtherdisruption of cell clusters as they enter the lipid separating unit suchthat desired non-lipid containing cells are generally dissociated fromthe adipocytes. The dispersing port is located proximal to the bottom ofthe lipid separating unit and the pores are directed downward such thatthe flow path of the cell mixture out of the dispersing head maximizesfluid shear applied to the cells.

The lipid separating unit is adapted for phase separation of lipids andlipid containing cells from a population of non-lipid containing cellsand includes a collection port disposed in the lipid separating unit tocollect the population of non-lipid containing cells from under thefloating lipid layer. When disposed in a unitary sterilizable apparatushaving closed flow path, collection of various cells from adiposecontaining tissue is provided without a need for centrifugation. In oneaspect, at least one filter is disposed in the fluid flow path betweenthe digestion chamber and the lipid separating unit. In one aspect,apparatus includes a digestion mesh having a pore size of about 1,000microns, at least one dispersing filter having a pore size of about 250microns and dispersing head having pores with a pore size of about 500microns.

The method of preparing a population of cells for cell transplantationcan include the following steps: a) dissociating a sample of donoradipose tissue into individual cells and small clusters of cells untilthe dissociated cells and clusters of cells are reduced in diameter toabout 1,000 microns or less, such as about 500 microns or less, or evenabout 250 microns or less; b) phase separating the individual cells andsmall clusters of cells into an aqueous cellular layer and a lipid layerwithout centrifugation; and c) collecting cells (e.g., stromal vascularfraction cells or a reparative cell population of stem cells) for celltransplantation from the aqueous cellular layer.

More specifically, the method provided herewith for separating stemcells from tissue includes the steps of introducing a tissue into adigestion chamber that includes a digestion fluid and an internaldigestion mesh, recirculating the digestion fluid across the digestionmesh until the tissue is separated into a digestion mixture thatincludes individual cells and cell clusters, followed by phaseseparating the digestion mixture through an aqueous medium disposed in alipid separating unit. After the phase separation, wherein theconstituent cells of the digestion mixture are separated on the basis ofdensity in an aqueous medium, desired stem cell populations arecollected from below the floating lipids and lipid containing cellswithin the separating unit.

The stem cell preparations provided herein can provide a direct sourceof stromal cells, e.g., chondrocytes and/or chondrocyte progenitorcells, that are capable of migrating to the defected site, attachingthereto, and elaborating cartilage-specific macromolecules andextracellular matrix proteins for the production of new cartilage at thedefect site. The stem cell preparation provided herein may also providecells that produce biological factors that promote chondrogenesis andthe migration of cells such as chondrogenic stem cells or chondrocytes,from the in vivo environment, including from theperiosteal/perichondrial tissue, adjacent to the defect site, and/ordifferentiation thereon and therein.

The stem cell preparation provided herein may include chondrocytes,chondrocyte progenitor cells including mesenchymal stem cells,fibroblasts, fibroblast-like cells and/or cells capable of producingcollagen type II and other collagen types, and proteoglycans which aretypically produced in cartilaginous tissues. The stem cells can beobtained from the patient (or subject) or a histocompatible donor. Thechondrocytes, progenitor cells, fibroblast-like cells and other cellsand/or elements that comprise the stroma may be fetal or adult inorigin, and may be derived from convenient sources such as cartilage,bone, skin, ligaments, tendons, muscles, placenta, umbilical cord, etc.For example, stromal cells such as chondrocytes may be derived from anytype of cartilage, including but not limited to, hyaline cartilage,costal cartilage, fibrous cartilage, etc., which can be obtained bybiopsy (where appropriate) or upon autopsy. Chondrocyte progenitor cellsmay be derived from various sources including bone marrow, periosteum,perichondrium or various sources of undifferentiated human mesenchyme.Fibroblasts can be obtained in quantity rather conveniently fromforeskin, preferably fetal foreskin, or, alternatively, any appropriatecadaver organ. Fetal cells, including fibroblast-like cells andchondrocyte progenitor cells, may be obtained from umbilical cord orplacenta tissue or umbilical cord blood. Although stem cells from avariety of sources may be used in the prevent invention, the stem cellscan be derived from cells of fetal origin which may be viewed as“universal donors” so as to minimize the risk of immunologicalrejection. Alternatively, obtaining stem cells from mature adult sourcesis also very useful, particularly for autologous transplantation.

The stromal cells may be readily isolated by disaggregating anappropriate tissue which is to serve as the source of the cells. Thismay be readily accomplished using techniques known to those skilled inthe art. For example, the tissue can be disaggregated mechanicallyand/or treated with digestive enzymes and/or chelating agents thatweaken the connections between neighboring cells making it possible todisperse the tissue into a suspension of individual cells withoutappreciable cell breakage. Enzymatic dissociation can be accomplished bymincing the tissue and treating the minced tissue with any of a numberof digestive enzymes either alone or in combination. These include butare not limited to trypsin, chymotrypsin, collagenase, elastase, and/orhyaluronidase, Dnase, pronase, etc. Mechanical disruption can also beaccomplished by a number of methods including, but not limited to theuse of grinders, blenders, sieves, homogenizers, pressure cells, orsonicators to name but a few. For a review of tissue disaggregationtechniques, see Freshney, Culture of Animal Cells. A Manual of BasicTechnique, latest edition, A. R. Liss, Inc., New York, 1987, Ch. 9.

Once the tissue has been reduced to a suspension of individual cells,the suspension can be fractionated into subpopulations from whichchondrocytes, fibroblasts and/or other stromal cells and/or elements canbe obtained. This also may be accomplished using standard techniques forcell separation including but not limited to cloning and selection ofspecific cell types, selective destruction of unwanted cells (negativeselection), separation based upon differential cell agglutinability inthe mixed population, freeze-thaw procedures, differential adherenceproperties of the cells in the mixed population, filtration,conventional and zonal centrifugation, centrifugal elutriation(counter-streaming centrifugation), unit gravity separation, countercurrent distribution, electrophoresis and fluorescence-activated cellsorting. For a review of clonal selection and cell separationtechniques, see Freshney, supra, Ch. 11 and 12.

For example, the isolation of chondrocytes, chondrocyte progenitors,fibroblasts or fibroblast-like cells is carried out as follows: freshhuman cartilage tissue can be thoroughly washed and minced in Hanksbalanced salt solution (HBSS) in order to remove serum. The mincedtissue is incubated from 1-12 hours in a freshly prepared solution of adissociating enzyme such as trypsin. After such incubation, thedissociated cells are suspended, pelleted by centrifugation and platedonto culture dishes. All fibroblasts will attach before other cells,therefore, appropriate stromal cells can be selectively isolated andgrown. The isolated stromal cells can then be grown to confluency,lifted from the confluent culture and administered to the cartilagedefect site in vivo (see, e.g., Naughton et al., 1987, J. Med.18(3&4):219-250). Fibroblast-like cells may also be isolated from humanumbilical cords (33-44 weeks). Fresh tissues may be minced into piecesand washed with medium or snap-frozen in liquid nitrogen until furtheruse. The umbilical tissues may be disaggregated as described above.

Once chondrocytes or chondrocyte progenitor cells have been isolated,their population can be expanded mitotically in vitro in order to obtainthe cell preparation for administration. Methods for the selection ofthe most appropriate culture medium, medium preparation, and cellculture techniques are well known in the art and are described in avariety of sources, including Doyle et al., (eds.), 1995, Cell & TissueCulture: Laboratory Procedures, John Wiley & Sons, Chichester; and Hoand Wang (eds.), 1991, Animal Cell Bioreactors, Butterworth-Heinemann,Boston.

The cells can be transferred or “passaged” to fresh medium when theyreach an appropriate density, such as 3 to 6.5×10⁴/cm², or, for example,when they reach a defined percentage of confluency on the surface of aculture dish. During incubation, the stromal cells may stick to thewalls of the culture vessel where they can continue to proliferate andform a confluent monolayer. This can be prevented or minimized, forexample, by transferring a portion of the cells to a new culture vesselhaving fresh medium, since the presence of a confluent monolayer in theculture vessel will tend to “shut down” the growth of cells in theculture. Removal of the confluent monolayer or transfer of a portion ofthe cells to fresh media in a new vessel will usually restoreproliferative activity of the cells. Such removal or transfer should bedone in any culture vessel which has a monolayer exceeding about 25%confluency. Alternatively, the liquid culture can be agitated, forexample, on an orbital shaker or in roller bottles, to prevent orminimize the cells from sticking to the vessel walls.

In addition, once the stromal cells have been established in culture,they may be maintained or stored in cell “banks” comprising eithercontinuous in vitro cultures of cells requiring regular transfer, orcells which have been cryopreserved. Cryopreservation of the cells maybe carried out according to known methods, such as those described inDoyle et al., 1995, supra. Cryoprotective agent and its use incryopreservation of cellular matter is disclosed in U.S. Pat. No.5,071,741 to Brockbank (the entire contents of which is incorporatedherein by reference). Furthermore, compositions and methods forculturing and freezing cells and tissues are also disclosed in U.S. Pat.No. 5,102,783 to Alkemade et al. (the entire contents of which isincorporated herein by reference).

For example, but not by way of limitation, cells may be suspended in a“freeze medium” such as, for example, culture medium further comprising20% FBS and 9% dimethylsulfoxide (DMSO), with or without 5-10% glycerol,at a density, for example, of about 4-10×10⁶ cells/ml. The cells aredispensed into glass or plastic ampoules (Nunc) which are then sealedand transferred to the freezing chamber of a programmable freezer. Theoptimal rate of freezing may be determined empirically. For example, afreezing program that gives a change in temperature of −1° C./minthrough the heat of fusion may be used. Once the ampoules have reached−180° C., they are transferred to a liquid nitrogen storage area.Cryopreserved cells can be stored for a period of years, though theyshould be checked at least every 5 years for maintenance of viability.

The cryopreserved cells constitute a bank of cells, portions of whichcan be “withdrawn” by thawing and then used to produce new cartilagetissue as needed. Thawing should generally be carried out rapidly, forexample, by transferring an ampoule from liquid nitrogen to a 37° C.water bath. The thawed contents of the ampoule should be immediatelytransferred under sterile conditions to a culture vessel containing anappropriate medium such as RPMI 1640 conditioned with 10% FBS and 5% ES.It is advisable that the cells in the culture medium be adjusted to aninitial density of about 3-6×10⁵ cells/ml so that the cells cancondition the medium as soon as possible, thereby preventing aprotracted lag phase. Once in culture, the cells may be examined daily,for example, with an inverted microscope to detect cell proliferation,and subcultured as soon as they reach an appropriate density.

In addition to chondrocytes, chondrocyte progenitors, fibroblasts orfibroblast-like cells, other cells which aid in the production ofdesired cells may be added to the stem cell preparation foradministration. For example, other cells found in loose connectivetissue may be seeded along with chondrocytes or fibroblasts. Such cellsinclude but are not limited to endothelial cells, pericytes,macrophages, monocytes, plasma cells, mast cells, adipocytes, etc. Thesestromal cells may readily be derived from appropriate organs includingumbilical cord or placenta or umbilical cord blood using methods knownin the art such as those discussed above.

Moreover, the stem cell preparation may further comprise one or moreother components, including selected extracellular matrix components,such as one or more types of collagen known in the art, as well asgrowth factors and/or drugs. Growth factors which may be usefullyincorporated into the stem cell preparation include one or more tissuegrowth or stimulatory factors known in the art or to be identified inthe future, including but not limited to any member of the TGF-β family,BMPs that stimulate cartilage formation, e.g., BMP-2, BMP-12, andBMP-13, factors that stimulate migration of stromal cells and/or matrixdeposition, insulin-like growth factor (IGF)-I and -II, fibroblastgrowth factor (FGF), Actavin, growth hormone, etc.

Drugs which may be usefully incorporated into the stem cell preparationinclude anti-inflammatory compounds such as non-steroidalanti-inflammatories, immunosuppressants such as the cyclosporins, aswell as local anesthetics. Other components may also be included in thestem cell preparation, including but not limited to any of thefollowing: (1) buffers to provide appropriate pH and isotonicity; (2)lubricants; (3) viscous materials to retain the cells at or near thesite of administration, including, for example, alginates, agars andplant gums; and (4) other cell types that may produce a desired effectat the site of administration, such as, for example, enhancement ormodification of the formation of cartilage tissue or its physicochemicalcharacteristics, or support for the viability of the cells, orinhibition of inflammation or rejection.

According to one embodiment of the invention, growth regulatory orstimulatory factors including, but not limited to, TGF-β and ascorbate,or BMPs that stimulate cartilage formation such as BMP-2, BMP-12, andBMP-13 may be added to the administration site before, during or afteradministration of the stem cell preparation in order to promote theproduction of new cartilage at the site. Moreover, such growthregulatory factors can be administered to the site at the time ofadministration of the stromal cells, either as a separate preparationor, as noted supra, as part of the stromal cell preparation.

In addition, the stromal cells may be genetically engineered to producegrowth factors such as TGF-β, as well as other biological factors suchas factors that stimulate chondrogenesis or the migration ofchondrogenic and other stromal cells to the defected site. The stromalcells as provided herein can be genetically engineered to produce geneproducts that promote the successful production or repair of cartilageat a defect site and/or for use in gene therapies. For example, thestromal cells can be genetically engineered to express anti-inflammatoryfactors, e.g., anti-GM-CSF, anti-TNF, anti-IL-1, anti-IL-2, etc., toreduce the risk of rejection or degenerative changes in the cartilagedue to rheumatoid disease or other inflammatory reactions. Also, thestromal cells can be genetically engineered to express peptides orpolypeptides corresponding to the idiotype of neutralizing antibodiesfor granulocyte-macrophage colony stimulating factor (GM-CSF), TNF,IL-1, IL-2, or other inflammatory cytokines. IL-1 has been shown todecrease the synthesis of proteoglycans and collagens type II, IX, andXI (Tyler et al., 1985, Biochem. J. 227:869-878; Tyler et al., 1988,Coll. Relat. Res. 82:393-405; Goldring et al., 1988, J. Clin. Invest.82:2026-2037; and Lefebvre et al., 1990, Biophys. Acta. 1052:366-372)and is a potent stimulator of cartilage resorption and of the productionof inflammatory mediators by chondrocytes (Campbell et al., 1991, J.Immunol. 147:1238-1246). TNF also inhibits synthesis of proteoglycansand type II collagen, although it is much less potent than IL-1 (Yaron,I., et al., 1989, Arthritis Rheum. 32:173-180; Ikebe, T., et al., 1988,J. Immunol. 140:827-831; and Saklatvala, J., 1986, Nature 322:547-549).Once the genetically engineered stromal cells are administered into anindividual, the presence of the anti-inflammatory gene products canbring about amelioration of immunological rejection or the inflammatoryreactions associated with rheumatoid or joint disease.

In another embodiment, the stromal cells can be genetically engineeredto express a gene which would exert a therapeutic effect, e.g., in theproduction of TGF-β to stimulate cartilage production, or other factorssuch as BMP-13 to promote chondrogenesis and/or prevent bone formationor stimulatory factors that promote migration of stromal cells and/ormatrix deposition. In addition, the stromal cells can be geneticallyengineered to express a gene for which a patent is deficient. Forexample, genes that prevent or ameliorate symptoms of various types ofrheumatoid or joint diseases may be underexpressed or down-regulatedunder disease conditions. Specifically, expression of genes involved inpreventing inflammatory reactions in rheumatoid or joint diseases may bedown-regulated.

Alternatively, the activity of gene products may be diminished, leadingto the manifestations of some or all of the above pathologicalconditions and eventual development of symptoms of rheumatoid or jointdiseases. Thus, the level of gene activity may be increased by eitherincreasing the level of gene product present or by increasing the levelof the active gene product present at the defect site. By administeringstromal cells genetically engineered to express the active target geneproduct into the defect site of a rheumatoid or joint disease patientwho is deficient for that product, the level of the target gene productand/or the activity of that product can be modulated to prevent orameliorate the symptoms of rheumatoid or joint diseases. “Target gene,”as used herein, refers to a gene involved in rheumatoid or jointdiseases in a manner by which modulation of the level of target geneexpression or of target gene product activity may act to amelioratesymptoms of rheumatoid or joint diseases by preventing resorption ofcartilage and production of inflammatory mediators by chondrocytes.

In addition, patients may be treated by gene replacement therapy byadministering the stromal cells provided herein. Thus, replacement orrepaired cartilage may be designed specifically to meet the requirementsof an individual patient; for example, the stromal cells may begenetically engineered to regulate one or more genes; or the regulationof gene expression may be transient or long-term; or the gene activitymay be non-inducible or inducible.

The stromal cells provided herein can also be genetically engineered to“knock out” expression of factors that promote inflammation or rejectionat the administration site. Negative modulatory techniques for thereduction of target gene expression levels or target gene productactivity levels are discussed below. “Negative modulation”, as usedherein, refers to a reduction in the level and/or activity of targetgene product relative to the level and/or activity of the target geneproduct in the absence of the modulatory treatment. The expression of agene native to stromal cell can be reduced or knocked out using a numberof techniques, for example, expression may be inhibited by inactivatingthe gene completely (commonly termed “knockout”) using standardhomologous recombination techniques. Usually, an exon encoding animportant region of the protein (or an exon 5′ to that region) isinterrupted by a positive selectable marker (for example neo),preventing the production of normal mRNA from the target gene andresulting in inactivation of the gene. A gene may also be inactivated bycreating a deletion in part of a gene, or by deleting the entire gene.By using a construct with two regions of homology to the target genethat are far apart in the genome, the sequences intervening the tworegions can be deleted. Mombaerts et al., 1991, Proc. Nat. Acad. Sci.U.S.A. 88:3084-3087.

Antisense and ribozyme molecules which inhibit expression of the targetgene can also be used to reduce the level of target gene activity. Forexample, antisense RNA molecules which inhibit the expression of majorhistocompatibility gene complexes (HLA) have been shown to be mostversatile with respect to immune responses. Furthermore, appropriateribozyme molecules can be designed as described, e.g., by Haseloff etal., 1988, Nature 334:585-591; Zaug et al., 1984, Science 224:574-578;and Zaug and Cech, 1986, Science 231:470-475. Still further, triplehelix molecules can be utilized in reducing the level of target geneactivity. These techniques are described in detail by L. G. Davis etal., eds, Basic Methods in Molecular Biology, 2nd ed., Appleton & Lange,Norwalk, Conn. 1994.

Using any of the foregoing techniques, the expression of IL-1 can beknocked out in the chondrocytes to reduce the risk of resorption ofcartilage and production of inflammatory mediators by the chondrocytes.Likewise, the expression of MHC class II molecules can be knocked out inorder to reduce the risk of rejection of the implant.

Methods that may be useful to genetically engineer the cells arewell-known in the art. For example, a recombinant DNA construct orvector containing the gene of interest may be constructed and used totransform or transfect the stromal cells provided herein. Suchtransformed or transfected cells that carry the gene of interest, andthat are capable of expressing said gene, are selected and clonallyexpanded in culture. Methods for preparing DNA constructs containing thegene of interest, for transforming or transfecting cells, and forselecting cells carrying and expressing the gene of interest arewell-known in the art. See, for example, the techniques described inManiatis et al., 1989, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates & Wiley Interscience, N.Y.; and Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

The cells can be engineered using any of a variety of vectors including,but not limited to, integrating viral vectors, e.g., retrovirus vectoror adeno-associated viral vectors; or non-integrating replicatingvectors, e.g., papilloma virus vectors, SV40 vectors, adenoviralvectors; or replication-defective viral vectors. Where transientexpression is desired, non-integrating vectors and replication defectivevectors can also be used, since either inducible or constitutivepromoters can be used in these systems to control expression of the geneof interest. Alternatively, integrating vectors can be used to obtaintransient expression, provided the gene of interest is controlled by aninducible promoter. Other methods of introducing DNA into cells includethe use of liposomes, lipofection, electroporation, a particle gun, orby direct DNA injection.

Hosts cells are preferably transformed or transfected with DNAcontrolled by, i.e., in operative association with, one or moreappropriate expression control elements such as promoter or enhancersequences, transcription terminators, polyadenylation sites, amongothers, and a selectable marker. Following the introduction of theforeign DNA, engineered cells may be allowed to grow in enriched mediaand then switched to selective media. The selectable marker in theforeign DNA confers resistance to the selection and allows cells tostably integrate the foreign DNA as, for example, on a plasmid, intotheir chromosomes and grow to form foci which, in turn, can be clonedand expanded into cell lines. This method can be advantageously used toengineer cell lines which express the gene product.

Any promoter may be used to drive the expression of the inserted gene.For example, viral promoters include, but are not limited to, the CMVpromoter/enhancer, SV40, papilloma virus, Epstein-Barr virus, elastingene promoter and β-globin. The control elements used to controlexpression of the gene of interest should allow for the regulatedexpression of the gene so that the product is synthesized only whenneeded in vivo. If transient expression is desired, constitutivepromoters are used in a non-integrating and/or replication-defectivevector. Alternatively, inducible promoters could be used to drive theexpression of the inserted gene when necessary. Inducible promoters canbe built into integrating and/or replicating vectors. For example,inducible promoters include, but are not limited to, metallothionien andheat shock protein.

A variety of methods may be used to obtain the constitutive or transientexpression of gene products engineered into the stromal cells. Thestromal cells can be engineered to express such gene productstransiently and/or under inducible control during the post-operativerecovery period, or as a chimeric fusion protein anchored to the stromalcells, for example, as a chimeric molecule composed of an intracellularand/or transmembrane domain of a receptor or receptor-like molecule,fused to the gene product as the extracellular domain. Once the stromalcells have been genetically engineered, they may be administered to thepatient to produce new cartilage in cases of degenerative connectivetissue disease or trauma.

Formulations and Administration

Both glycosaminoglycan composition and the stem cells provided hereincan be formulated into any suitable pharmaceutically acceptablepreparations and/or formulations for administration. For parenteralproducts, the carrier will usually comprise sterile water, althoughother ingredients may be included, e.g., to aid solubility or forpreservation purposes. Injectable suspensions may also be prepared, inwhich case appropriate liquid carriers, suspending agents, and the likemay be employed.

In alternative embodiments, the glycosaminoglycan composition describedherein can be in sterile solution, suspension, or other pharmaceuticallyacceptable formulations. The glycosaminoglycan composition describedherein can be applied directly to the affected connective tissue, or isadapted for intra-articular and/or systemic or parenteraladministration. Systemic administrations can include, but are notlimited to, intramuscular, intravenous or subcutaneous injection. Thus,in one embodiment, the glycosaminoglycan composition described hereinwhich has been specially adapted for intra-articular use and/orparenteral (e.g., intravenous or intramuscular) are sterile solutions orsuspensions comprised of therapeutic amounts of chondroitin sulfate,N-acetyl D-glucosamine, and hyaluronan. In addition to theafore-mentioned active agents, it can be appreciated by one of skill inthe art that the glycosaminoglycan compositions described herein whichare adapted for intra-articular use, and other therapeutic uses, canalso comprise preservatives, pharmaceutically active carriers,excipients, stabilizers, buffers, antimicrobial growth inhibitors andthe like, and the use of such is contemplated by the invention.

It is also contemplated that other formulations for theglycosaminoglycan composition provided herein are also possible and arewithin the scope of the invention, e.g., a powdered formulation suitablefor reconstitution with a suitable injectable liquid or for addition toa pre-selected lavage fluid. In particular, it can be appreciated by oneof skill in the art that the active agents of the glycosaminoglycancomposition can be stored in a freeze dried or lyophilized state forreconstitution and use at a desired time.

In yet another embodiment, the glycosaminoglycan composition describedherein is attached to an impregnated bolus or a sheet of materialadapted for implantation directly onto or between connective tissues ofa mammalian body, for example to prevent the formation of post-operativeadhesions, e.g., scar tissue formation. The glycosaminoglycancomposition described herein can be impregnated into an absorptivegauze-like material or coated onto the material or joined to thematerial by adhesion and/or capillary action to a mesh or gauze. Thematerial onto which the glycosaminoglycan composition is attached orabsorbed may be either a permanent implant or it may be biodegradable.In one embodiment, the therapeutic glycosaminoglycan composition can bedispersed on or within a malleable material which can be shaped forinsertion into diseased or excised tissue spaces. In yet anotherembodiment, the glycosaminoglycan composition described herein can beattached to a bandage or other surgical materials, including but notlimited to surgical suture material, surgical staple, or a device suchas a buckle. Surgically implanted devices and sheet of materials havingdrugs/compositions attached thereon are disclosed in U.S. Pat. No.6,534,693 (the entire contents of which is incorporated herein byreference).

The stem cell preparations provided herein can be administered eitherbefore, during (as an admixture or generally concurrently), or afteradministration of the glycosaminoglycan composition. For example, thestem cells and/or the stem cell preparations can be seeded into thedefect site before, during or after administration of theglycosaminoglycan composition, e.g., by injection into, and/or directapplication to the defect site. When the stem cells are injected intothe site, such injection can be achieved by any means that maintains theviability of the cells, e.g., via syringe or more preferably, via anarthroscope. According to an embodiment of the present invention, thenumber of stem cells administered can range from approximately 1×10⁶ to30×10⁶ stem cells. In yet another embodiment, the stem cell preparationsprovided herein can also be mixed with the glycosaminoglycan compositionprovided herein immediately before the administration to the defectsite.

According to an embodiment of the present invention, the stem cells tobe seeded are surgically obtained from a subject, e.g., from adiposetissue, cartilage and/or bone marrow, in a separate surgical procedure,isolated, potentially cultured in vitro, to obtain an appropriate amountof cells and administered to the subject. The stem cells provided hereincan form a stromal matrix that resembles the in vivo microenvironment ofcartilage tissue, allowing for the production of new cartilage at thedefect site. The stem cells administered to the site provide importantbiological factors that promote chondrogenesis and the migration ofstromal cells, thus promoting the production of a living stromal tissuethat provides the support, growth factors, and regulatory factorsnecessary to sustain long-term active proliferation of the stromal cellsin vivo. The stem cells provided herein may additionally provide factorsthat promote the deposition of the living stromal matrix at the defectsite. The proliferating cells mature and segregate properly within thematrix to form new cartilage tissue at the defect site in vivo.

The successful repair or replacement of damaged cartilage can beenhanced if the new cartilage tissue can be fixed in place at the siteof repair. Post-implantation movement may cause the new cartilage tissueto become dislodged from the site if a pro-active fixation technique isnot employed. Various methods can be used to fix the new cartilagetissue in place, including: patches derived from a bioresorbable polymeror biocompatible tissues, which can be placed over the site and sutured;bioabsorbable sutures or other fasteners, e.g., pins, staples, tacks,screws, anchors, glues, e.g., fibrin glue; non-absorbable fixationdevices, e.g., sutures, pins, screws and anchors; adhesives; and the useof interference fit geometries.

Treatment and/or Repair of Connective Tissue Damage

It is contemplated by the present invention that the pharmaceuticalpreparations comprising the glycosaminoglycan composition and stem cellsor the stem cell preparation described herein demonstrate enhancedeffectiveness in the treatment of primary or secondary damage and injuryto any connective tissues in humans and animals. Such diseases andinjuries include, but are not limited to, arthritic diseases,osteoarthritis (OA), rheumatoid arthritis (RA), osteochondrosisdessicans (OCD), cartilage damage, joint injuries, joint inflammation,joint synovitis, degenerative joint disease (DJD), post surgical DJD,traumatic injuries, fractures, tendon damage, ligament damage, skeletaldamage, musculoskeletal damage, bone damage, fiber damage, adiposetissue damage, blood cell damage, and plasma damage, and the like.

In one embodiment of the invention, a pharmaceutical preparationcomprising a glycosaminoglycan composition in combination with stemcells or a stem cell preparation, and methods of using suchpharmaceutical preparation described herein for the treatment ofrheumatoid arthritis (RA) are provided. Rheumatoid arthritis is thoughtto be a human autoimmune disease characterized by chronic inflammationof the synovial joints and progressive erosion of the articularcartilage matrix. Although the etiology and pathology of rheumatoidarthritis is not totally understood, certain parameters are almostalways present. Cytokines, free radicals, reactive oxygen species anddegrading enzymes are formed as a result of phagocytic activity in therheumatoid arthritis affected joint. Inflammatory mediators such as IL1,TNF, MMPs, RNOs have long been implicated as mediators of articulardamage in rheumatoid arthritis as well as low molecular weight HA. Thebiological response to the presence of these products in rheumatoidarthritis joints may explain the increase in GAG levels present as thebody's defense mechanisms become overwhelmed as the disease progresses.

While not wishing to be bound by any particular theories of activity, itis postulated herein that the body's normal response to rheumatoidarthritis may not be effective in controlling its pathology, and anendogenous increase of specific GAGs, as provided by the compositions ofthe invention, will result in a more favorable long term response totreatment of rheumatoid arthritis. The present invention provides thatadministration of a pharmaceutical preparation comprising aglycosaminoglycan composition comprising one or more GAGs, or homologsor anologs thereof, in combination with stem cells or a stem cellpreparation, as set forth herein, will reduce the production andpresence of the aforementioned inflammatory mediators via specificbiological and chemical pathways, and promote the production of a livingstromal tissue that provides the support, growth factors, and regulatoryfactors necessary to sustain long-term active proliferation of thestromal cells in vivo, thus, allowing production of new cartilage orother relevant connective tissues in the defect site.

Accordingly, the present invention provides methods of treatment forconnective tissue conditions including rheumatoid arthritis comprisingperiodic administrations of therapeutic amounts of the pharmaceuticalpreparation comprising a glycosaminoglycan composition in combinationwith stem cells or a stem cell preparation, as described herein. It canbe appreciated by one of skill in the art that the treatment regimen(e.g., frequency of administration and dosage) will vary according tothe history, signalment, clinical stage and/or severity of therheumatoid arthritis disease in a particular subject. In certainembodiments of the invention, the pharmaceutical preparation of theinvention may be used in conjunction with other known rheumatoidarthritis treatment agents such as, e.g., DMARDs, TNF blockers, IL-1Ras,immunosuppressants and the like.

One method provided by the present invention is a first treatmentregimen comprising a pretreatment with, e.g., a suitable DMARD, TNFblocker, or other immunosuppressant in conjunction with thepharmaceutical preparation comprising a glycosaminoglycan compositionand stem cells or a stem cell preparation, or as a separatepre-treatment to provide a systemic short term down regulation of thedisease process, followed by a second regimen of treatment with thepharmaceutical preparation comprising a glycosaminoglycan compositionand stem cells or a stem cell preparation to normalize and stabilize thebody's response to alleviate the symptoms of rheumatoid arthritis on along term basis without the deleterious side effects of e.g., systemicimmunosuppressant agents such as TNF blockers. It is contemplated thatthe initial treatment regimen with, e.g., a suitable DMARD, TNF blockeror other immunosuppressant can be in conjunction with therapeuticamounts of the pharmaceutical preparation comprising a glycosaminoglycancomposition and stem cells or a stem cell preparation, as providedherein.

In yet another embodiment of the present invention the pharmaceuticalpreparation comprising a glycosaminoglycan composition and stem cells ora stem cell preparation, as set forth herein, can further comprise atherapeutically effective amount of other suitable therapeuticsincluding, but not limited to, antibiotics. For instance, suitableantibiotics for use in the compositions provided herein include, but arenot limited to any of the antibiotics that can be adapted forintra-articular use, (see, e.g., “Infectious Arthritis” Alicia L.Bertone, pp. 397-409, in Joint Disease in the Horse,” W. B. Sanders,1996 (ISBN 0-7216-5135-6)). As can be appreciated by one of skill in theart, the choice of antibiotics and other suitable therapeutics, andtheir therapeutically effective amounts, can depend many factorsincluding, but not limited to, e.g., the etiology of the infectiousorganism being treated or personal preference of the treatingveterinarian or physician.

The pharmaceutical preparation comprising a glycosaminoglycancomposition and stem cells or a stem cell preparation, as providedherein, can also further comprise or exclude other therapeutic agentsinsofar as it is generally used as a therapeutic for connective tissuedisease (e.g., tendonitis). Examples of other such therapeutic agentsinclude, but are not limited to, synthetic and non-syntheticcorticosteroid agents, nonsteroidal anti-inflammatory drugs,antirheumatics, immunoregulators, immunosuppressant, articular functionaugmenters, and interleukin production inhibitors. Specific examples ofcorticosteroid agents include, but are not limited to dexamethasone,hydrocortisone, triamcinolone, betamethasone, predonisolone,methylpredonisolone, halopredone, beclomethasone and the like. Specificexamples of non-steroidal anti-inflammatory agents include, but are notlimited to diclofenac, indomethacin, ibuprofen, ketoprofen, aspirin,diflunisal, fulfenamic acid, floctafenine, tolfenamic acid, sulindac,fenbufen, salicylic acid, acemetacin, proglumetacin, nabumetone,protizinic acid, thiaprofen, oxaprozin, loxoprofen, alminoprofen,zaltoprofen, flurbiprofen, flurbiprofen and the like.

In one embodiment, the pharmaceutical preparation comprising aglycosaminoglycan composition and stem cells or a stem cell preparation,as provided herein, can further comprise at least one pyrazolylbenzenesulfonamide compound, e.g., as set forth in U.S. Pat. No.5,756,529 and U.S. Pat. No. 5,466,823 (the contents of which areincorporated herein by reference). In particular, the pharmaceuticalpreparation comprising a glycosaminoglycan composition and stem cells ora stem cell preparation, as provided herein, can further comprise adiaryl substituted pyrazole useful for treatment of inflammation and/orpain. It is specifically contemplated that the pharmaceuticalpreparation comprising a glycosaminoglycan composition and stem cells ora stem cell preparation, as provided herein, can further comprisetherapeutic amounts of any of the class of diaryl substituted pyrazolestheir isomers, analogs and/or metabolites. In particular, thesecompounds reduce inflammation and/or pain primarily via inhibition ofcyclooxygenase-2 (COX-2). In an embodiment, the pharmaceuticalpreparation comprising a glycosaminoglycan composition and stem cells ora stem cell preparation, as provided herein, further comprises anon-steroidal agent that reduces inflammation and/or pain primarily viainhibition of cyclooxygenase-2 (COX-2) and with the substantial absenceof inhibition of cyclooxygenase-1 (COX-1). Examples of suitable diarylsubstituted pyrazoles for use in the pharmaceutical preparations of thepresent invention, include, but are not limited to, celecoxib, rofecoxiband the like.

Examples of other agents which may be added to the core pharmaceuticalpreparation comprising a glycosaminoglycan composition and stem cells ora stem cell preparation, as set forth herein, include axetil, piroxicam,tenoxicam, ampiroxicam, meloxicam, D-penicillamine, bucillamine, goldsodium thiomalate, auranofin, lobenzarit, salazosulfapyridine,methotrexate, cyclophosphamide, azathioprine, mizoribine, cyclosporinand the like.

In a particular embodiment, the present invention also provides apharmaceutical preparation comprising a glycosaminoglycan compositionand stem cells or a stem cell preparation, optionally with a suitableantioxidant or free radical scavenger. In one embodiment, thepharmaceutical preparation comprising a glycosaminoglycan compositionand stem cells or a stem cell preparation, as provided herein, canfurther comprise a therapeutic amount of suitable superoxide dismutase(SOD) or other antioxidant including, but not limited to, examples setforth in U.S. Pat. No. 6,127,356 to Crapo et al., the contents of whichare incorporated herein by reference.

The present invention further provides a method for treating connectivetissue damage in humans or in animals comprising administering to a manor animal in need thereof, a therapeutically effective amount of thepharmaceutical preparation comprising a glycosaminoglycan compositionand stem cells or a stem cell preparation, as provided herein. In oneembodiment, the pharmaceutical preparation comprising aglycosaminoglycan composition and stem cells or a stem cell preparation,as described herein, is directly applied to the affected connectivetissues. In another embodiment, the pharmaceutical preparationcomprising a glycosaminoglycan composition and stem cells or a stem cellpreparation, as provided herein, is adapted for intra-articular and/orsystemic or parenteral administration. Systemic administrations caninclude, but are not limited to, intramuscular, intravenous orsubcutaneous injection. The connective tissue damage referred hereininclude any primary or secondary diseases or injuries to the connectivetissues in humans and/or animals Such diseases or injuries include, butare not limited to, arthritic diseases, osteoarthritis (OA), rheumatoidarthritis (RA), osteochondrosis dessicans (OCD), cartilage damage, jointinjuries, joint inflammation, joint synovitis, degenerative jointdisease (DJD), post surgical DJD, traumatic injuries, fractures, tendondamage, ligament damage, skeletal damage, musculoskeletal damage, bonedamage, fiber damage, adipose tissue damage, blood cell damage, andplasma damage.

One skilled in the art will understand that, in a method for treatingdiseases of connective tissue, therapeutic dosage will vary according tothe specific condition being treated and the severity of the disease,etc., and, therefore, can be given in a single dose, and then repeatedas needed, or the dosage can be given incrementally in several smallerdosages. Thus, the pharmaceutical preparation of the present inventioncan be formulated such that the recommended therapeutic dose is achievedby the administration of a single dose or by the administration ofseveral smaller doses. In certain methods, the injection is givensystemically every 3-4 days to weeks, and the intralesional injectionmay be a single injection or a series of injections at, e.g., 3-weekintervals.

Kit

It is apparent to one skilled in the art that the pharmaceuticalpreparation comprising a glycosaminoglycan composition and stem cells orcomponents for producing a stem cell preparation, as provided herein,can be included in a commercial package together with instructions forits use against a disease of connective tissue. Thus, the presentinvention further provides a kit comprising one or more containerscomprising the glycosaminoglycan composition, the stem cells or thecomponents for isolating a stem cell preparation, and instructions foruse of the glycosaminoglycan composition in combination of the stemcells or the stem cell preparations for treating connective tissuedamages in man or in animals. The kit provided herein can also includeother separate containers for other drugs, agents, compounds havingdesired therapeutic effects, including, but not limited to, syntheticand non-synthetic corticosteroid agents, nonsteroidal anti-inflammatorydrugs, antirheumatics, immunoregulators, immunosuppressant, articularfunction augmenters, and interleukin production inhibitors.

The glycosaminoglycan composition, the stem cells or the stem cellpreparations provided herein, each of which provided in the kit can bestored in any suitable container, including, but not limited to,syringes and vials, and in various dosage units. Preferably, theglycosaminoglycan composition comprises the dosages for chondroitinsulfate and N-acetyl D-glucosamine from between about 0.5 grams to about1.5 grams per unit dose, respectively, and the dosage for hyaluronanfrom about 10 mg to about 50 mg per unit dose. Each component providedin the kit is formulated in sterile solutions, suspensions, or anypharmaceutically acceptable formulations, and stored in suitablecontainers, separately or in combination, in various dosage units.Preferably, the containers in the kit containing each of the componentprovided herein are disposable.

In one embodiment, the kit contains multiple preloaded syringes. Atleast one syringe is preloaded with a preselected volume of from betweenabout 2 and about 10 ml of the glycosaminoglycan composition describedherein in a sterile solution comprised of proportionate weight to volumeratios comprised of e.g., about 1 gram of chondroitin sulfate as amixture of about 40% CS4 and 60% CS6 chondroitin sulfate, about 1 gramof N-acetyl D-glucosamine, and about 20-40 mg but especially about 30 mgof hyaluronan (e.g., sodium hyaluronate) per unit dose of theglycosaminoglycan composition. The preloaded syringe volume will ofcourse vary depending upon the state of disease at the target tissue andspecies of animal, etc. The kit can also contain one or more separatesyringes preloaded with a desired amount of other therapeutics wellknown in the art. In yet another embodiment, the kit contains at leastthree separate containers, one contains chondroitin sulfate, such as CS4or CS6, or a mixture of 40% CS4 and 60% CS6, one contains N-acetylD-glucosamine, and one contains hyaluronan (e.g., sodium hyaluronate).The three components are mixed in a desired dosage unit before it isadministered.

In yet another embodiment, the kit provided herein contains a materialadapted for implantation onto or between tissues of a mammalian body andwith the pharmaceutical preparation comprising a glycosaminoglycancomposition and stem cells or a stem cell preparation attached thereon.The material can be in the form of malleable semi-solid substrate, or amesh or gauze-like carrier, and can be dissolvable or biodegradable. Thepharmaceutical preparation comprising a glycosaminoglycan compositionand stem cells or a stem cell preparation can be impregnated into thematerial or coated onto the material or joined to the material byadhesion and/or capillary action, such as to a mesh, gauze, orgauze-like material. Alternatively, the pharmaceutical preparationcomprising a glycosaminoglycan composition and stem cells or a stem cellpreparation is attached to the material that also includes, but is notlimited to, a bandage, a surgical suture or staple material, andsurgical device, such as buckle, suitable for a surgical process ordevice.

Furthermore, the kit provided herein includes instructions indicating amethod of use of the pharmaceutical preparation comprising aglycosaminoglycan composition and stem cells or a stem cell preparationfor treating any primary or secondary diseases or injuries to theconnective tissues in humans or animals Such diseases or injuriesinclude, but are not limited to, arthritic diseases, osteoarthritis(OA), rheumatoid arthritis (RA), osteochondrosis dessicans (OCD),cartilage damage, joint injuries, joint inflammation, joint synovitis,degenerative joint disease (DJD), post surgical DJD, traumatic injuries,fractures, tendon damage, ligament damage, skeletal damage,musculoskeletal damage, bone damage, fiber damage, adipose tissuedamage, blood cell damage, and plasma damage. Instructions are normallyin the form of a written material but are not limited to such.

Having discussed the pharmaceutical preparation comprising aglycosaminoglycan composition and stem cells or a stem cell preparation,as provided herein, and the method of use thereof, providing an enhancedeffectiveness for the treatment of connective tissue damage, it will bemore clearly perceived and better understood from the following specificexamples which are intended to provide examples of certain embodimentsand not limit the present invention.

EXAMPLES Example 1 Adipose Tissue-Derived Stem Cell Preparation

Stem cells were prepared using the Autologous Regenerative Cell System(ARC System) (InGeneron, Inc., Houston, Tex.), which is described indetail above.

Example 2 Treatment with Glycosaminoglycan Composition in Combinationwith Stem Cell Therapy Case 1:

“Hobbs” Jones, a 13 year-old male canine, had multiple surgeries in thepast to correct medial patella and secondary chondromalacia andosteoarthrosis/osteophytosis manifesting in progressive weight-bearinglameness on the left (L) side. Hobbs suffered from advanced degenerativejoint disease on the left (L) stifle.

Stem cell preparation: Falciform adipose tissue stem cells wereharvested and prepared as described above in Example 1. Fat was obtainedfrom a small abdominal incision at the umbilicus of the anesthetizedanimal. This falciform fat was utilized to obtain stem cells to injectinto the knee.

Treatment Procedure: 1 ml of the stem cell preparation combined with 1ml POLYGLYCAN® (hyaluronan, N-acetyl D-glucosamine and chondroitinsulfate) was injected intraarticularly at the left (L) stifle.SIMPLICEF® (200 mg) and TRAMADOL® (50 mg), were also administered one(1) tablet every 24 hours, and three (3) tablets every 12 hours,respectively. Hobbs was maintained on a normal diet, kept quiet withrestricted household activities, and leashed outside only when necessaryfor 7-10 days. The incision site was monitored for increased swellingand any discharge or licking/chewing at site. Hobbs was rechecked on afollow-up visit every two weeks.

Treatment Results: Definitive efficacy was obtained within one weekafter injection. No further improvements were observed, but sustainedbenefit remained for one month post injection.

Case 2:

“Shiloh” Helmey, a 9 year-old female German Shepherd, had previousbilateral tibial/fibular fractures four (4) years before the currenttreatment. She had recent plate removal and script infection on theright (R) tibia, and recent concurrent diagnosis of cranial cruciateligament tear. Shiloh suffered from degenerative joint disease on theright (R) stifle.

Stem cell preparation: Falciform adipose tissue harvested stem cellswere harvested and prepared as described above in Example 1. Fat wasobtained from a small abdominal incision at the umbilicus of theanesthetized animal. This falciform fat was utilized to obtain stemcells to inject into the knee.

Treatment Procedure: 1 ml of the stem cell composition combined with 1ml POLYGLYCAN® was injected at the right (R) stifle. SIMPLICEF® (250 mg)and TRAMADOL® (50 mg), were also administered one (1) tablet every 24hours, and four (4) tablets every 12 hours, respectively. Shiloh was onnormal diet, but kept quiet with restricted household activities andleashed outside only when necessary for 7-10 days. The incision site wasmonitored for increased swelling and any discharge or licking/chewing atsite. Shiloh was rechecked on a follow-up visit every week.

Treatment Results: Definitive efficacy was obtained within 10 days afterinjection. Sustained benefit remained for one month post injection.

Case 3:

“Shitake” Harrelson, a 13 year-old male Great Dane, had previous tibialplateau leveling osteotomy on the right (R) side three (3) years beforethe current treatment. Shitake also had previous prepuce/penisamputation with scrotal uresthrostomy for neoplasia. He was recentlydiagnosed with progressive cranial cruciate ligament tear, as well asdegenerative joint disease on the right(R) stifle.

Stem cell preparation: Falciform adipose tissue harvested stem cellswere prepared as described above in Example 1. Fat was obtained from asmall abdominal incision at the umbilicus of the anesthetized animal.This falciform fat was utilized to obtain stem cells to inject into theknee.

Treatment Procedure: 1 ml of the stem cell composition combined with 1ml POLYGLYCAN® was injected at the right (R) stifle. SIMPLICEF® (200 mg)and TRAMADOL® (50 mg), were also administered one (1) tablet every 24hours, and six (6) tablets every 12 hours, respectively. Shitake was onnormal diet, but kept quiet with restricted household activities andleashed outside only when necessary for 7-10 days. The incision site wasmonitored for increased swelling and any discharge or licking/chewing atsite. Shiloh was rechecked on a follow-up visit every week.

Treatment Results: Almost immediate benefits were noted, and sustainedfor three (3) weeks. Unfortunately, Shitake died due to complicationsfrom GI issues/aspiration pneumonia unrelated to the treatment.

1. A composition comprising: a glycosaminoglycan composition comprisingchondroitin sulfate, N-acetyl D-glucosamine, and hyaluronan; andisolated stem cells.
 2. The composition of claim 1, wherein saidchondroitin sulfate is a mixture of chondroitin 4-sulfate andchondroitin 6-sulfate.
 3. The composition of claim 1, wherein saidisolated stem cells are progenitors of connective tissue comprisingloose, dense regular or elastic connective tissue.
 4. The composition ofclaim 1, wherein said stem cells are isolated from adipose tissue. 5.The composition of claim 1, wherein said composition is cryopreserved.6. The composition of claim 1, wherein said glycosaminoglycancomposition consists essentially of chondroitin sulfate, N-acetylD-glucosamine, and hyaluronan.
 7. The composition of claim 1, adaptedfor parenteral administration.
 8. The composition of claim 1, dispersedonto or within a semi-solid or solid material adapted for implantationonto damaged tissues.
 9. The composition of claim 1, further comprisingone or more therapeutic agents selected from the group consisting of asynthetic or a non-synthetic corticosteroid agent, nonsteroidalanti-inflammatory agent, antirheumatic agent, immunoregulation agent,immunosuppressant agent, articular function augmentor agent, growthfactor, and interleukin production inhibitors.
 10. A kit comprising: a)one or more containers comprising a glycosaminoglycan compositioncomprising chondroitin sulfate, N-acetyl D-glucosamine, and hyaluronan;and b) instructions for obtaining stem cells from a mammal and using thestem cells in combination with the glycosaminoglycan composition.